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1. (WO2019041344) METHODS AND COMPOSITIONS FOR SINGLE-STRANDED DNA TRANSFECTION
Document

Description

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Claims

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Description

Title of Invention : METHODS AND COMPOSITIONS FOR SINGLE-STRANDED DNA TRANSFECTION

[0001]
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002]
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 777072000240SEQLIST. txt, date recorded: August 18, 2017, size: 269 KB) .

FIELD OF THE INVENTION

[0003]
The present invention relates to methods and compositions for transfection of a single-stranded DNA into a cell, which are useful for gene editing.

BACKGROUND OF THE INVENTION

[0004]
DNA is normally present in cells in a double-stranded conformation. The double-stranded structure protects genetic information in the DNA against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. Single-stranded DNA (ssDNA) is rarely found in mammalian cells, including transient situations such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. Single-stranded DNA binding proteins are present in cells to bind ssDNA in order to maintain genomic stability.
[0005]
Argonautes are a family of nucleic acid-binding proteins ubiquitously found in the cells of bacteria, plants, archaea and animals. Argonautes are most well-known for their role in RNA interference (RNAi) . Argonaute proteins from most species bind to noncoding oligonucleotide RNAs, which serve as guide RNAs for the Argonaute proteins to recognize target mRNAs via sequence complementarity, and subsequently induce mRNA degradation or translational repression thereby inhibiting gene translation. Recently, Argonautes from several bacterial and archaeal species have been shown to bind single-stranded DNAs (ssDNAs) and use the ssDNAs as guides to cleave target DNA in a sequence-specific manner. In particular, Natronobacterium gregoryi Argonaute ( “NgAgo” ) has potent DNA-guided DNA cleavage activity at physiological temperatures, which makes NgAgo a suitable enzyme for in-cell gene editing applications. However, transfection of single-stranded guide DNA represents a challenging step for successful gene editing by the NgAgo system in cells.
[0006]
The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.
[0007]
BRIEF SUMMARY OF THE INVENTION
[0008]
The present application provides methods, compositions, and kits for transfecting a single-stranded DNA (ssDNA) into a cell, and for modifying a target nucleic acid in a cell using a DNA-guided nuclease (such as an Argonaute protein) and a single-stranded guide DNA. Exemplary modifications to the target nucleic acid include, but are not limited to, site-specific cleavage, introduction of mutations, insertion of exogenous sequences, sequence substitutions, and alteration of gene expression.
[0009]
One aspect of the present application provides a method of modifying a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) and a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA, wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of the contacting. In some embodiments, the cell is at log growth phase at the time of the contacting. In some embodiments, the cell has a confluency of about 30%-80%at the time of the contacting.
[0010]
One aspect of the present application provides a method of modifying a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) and a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA, wherein the medium has no more than about 2% (v/v) of serum. In some embodiments, the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the medium is essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of the contacting. In some embodiments, the cell is at log growth phase at the time of the contacting. In some embodiments, the cell has a confluency of about 30%-80%at the time of the contacting.
[0011]
One aspect of the present application provides a method of modifying a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) into the cell; and subsequently (b) transfecting a single-stranded guide DNA into the cell, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, the single-stranded guide DNA is transfected into the cell about 2 to about 48 hours (such as about 6-18 hours) after the transfection of the vector encoding the Ago protein. In some embodiments, step (b) comprises contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000, DEAE-dextran, or HD) . In some embodiments, the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0012]
In some embodiments according to any one of the methods described above, the DNA-guided nuclease is an Argonaute (Ago) protein. In some embodiments, the Ago protein cleaves the target locus. In some embodiments, wherein the target locus is a double-stranded DNA, the Ago protein induces a double-strand break in the target locus. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, the Ago protein is derived from Pedobacter heparinus. In some embodiments, the Ago protein is derived from Microcystis sp. In some embodiments, the Ago protein is derived from Microcystis aeruginosa. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any one of 85%, 90%, 95%, 98%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any one of 85%, 90%, 95%, 98%or more sequence homology, or about 100%sequence identity) to SEQ ID NO: 1. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any one of 85%, 90%, 95%, 98%or more sequence homology, or about 100%sequence identity) to SEQ ID NO: 2. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any one of 85%, 90%, 95%, 98%or more sequence homology, or about 100%sequence identity) to SEQ ID NO: 11. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any one of 85%, 90%, 95%, 98%or more sequence homology, or about 100%sequence identity) to SEQ ID NO: 41.
[0013]
In some embodiments according to any one of the methods described above, the sequence of the target locus comprises no more than about 3 (such as any one of 3, 2, 1, or 0) mismatches to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60% (such as at least about any one of 60%, 65%, 70%, 75%, 80%or more) . In some embodiments, the guide DNA is phosphorylated at the 5’ terminus. In some embodiments, the guide DNA is about 10 to about 50 nucleotides (nt) long, such as about 15 nt to about 35 nt, about 20 nt to about 27 nt, or about 23 nt to 25 nt.
[0014]
In some embodiments according to any one of the methods described above, the contacting is in the presence of a divalent metal ion, such as Mg 2+. In some embodiments, the concentration of the divalent metal ion is at least about 0.1 mM.
[0015]
In some embodiments according to any one of the methods described above, the cell is transfected with the guide DNA for at least two times. In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the DNA-guided nuclease (such as the Ago protein) is at least about 100: 1. In some embodiments, the nucleic acid encoding the DNA-guided nuclease (such as the Ago protein) is operably linked to a promoter. In some embodiments, the nucleic acid encoding the DNA-guided nuclease (such as the Ago protein) is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid. In some embodiments, the nucleic acid encoding the DNA-guided nuclease (such as the Ago protein) is an mRNA. In some embodiments, the nucleic acid encoding the DNA-guided nuclease (such as the Ago protein) is codon-optimized for the organism from which the cell is derived. In some embodiments, the DNA-guided nuclease (such as the Ago protein) comprises a nuclear localization signal (NLS) .
[0016]
In some embodiments according to any one of the methods described above, the method comprises treating the cell with one or more antibiotics. In some embodiments, the cell is free from contamination by non-viral microorganisms, such as mycoplasma.
[0017]
In some embodiments according to any one of the methods described above, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is a genomic DNA. In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is a viral DNA. In some embodiments, the target nucleic acid is integrated in the genome of the cell. In some embodiments, the target nucleic acid is not integrated in the genome of the cell.
[0018]
In some embodiments according to any one of the methods described above, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is a yeast cell, a fungus cell, or a plant cell. In some embodiments, the cell is derived from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the cell is an immune cell.
[0019]
In some embodiments according to any one of the methods described above, the modifying comprises site-specific cleavage of the target nucleic acid. In some embodiments, the modifying comprises introducing a mutation at the target locus selected from an insertion, a deletion, and a frameshift mutation.
[0020]
In some embodiments according to any one of the methods described above, wherein the target nucleic acid is present in a cell, the method further comprises contacting the target nucleic acid with a donor DNA comprising a sequence homologous to the sequence of the target locus under a condition that allows integration of the donor DNA at the target locus. In some embodiments, the method comprises transfecting the donor DNA into the cell. In some embodiments, the donor DNA is transfected into the cell separately from the guide DNA. In some embodiments, the donor DNA is transfected into the cell at least about 6 hours after the transfection of the guide DNA. In some embodiments, the donor DNA and the guide DNA are transfected into the cell simultaneously, and wherein the donor DNA and the guide DNA are separately packed with a transfection agent. In some embodiments, the donor DNA encodes a selection marker, such as a reporter protein. In some embodiments, the method further comprises assessing the cell for expression of the selection marker. In some embodiments, the modifying comprises knocking in an exogenous sequence at the target locus, wherein the donor DNA comprises the exogenous sequence. In some embodiments, the modifying comprises introducing a substitution mutation at the target locus, wherein the donor DNA comprises the substitution mutation. In some embodiments, the substitution mutation is a single nucleotide substitution. In some embodiments, the target locus is a disease-associated locus.
[0021]
In some embodiments according to any one of the methods described above, the method further comprises sequencing the target nucleic acid after the modifying.
[0022]
Insome embodiments according to any one of the methods described above, the modifying comprises inducing a phenotypic change to the cell. In some embodiments, the method further comprises assessing the phenotypic change to the cell. In some embodiments, the modifying comprises altering expression of the target nucleic acid. In some embodiments, the modifying comprises introducing a knockout mutation at the target locus. In some embodiments, the target locus is a disease-associated locus.
[0023]
One aspect of the present application provides a kit comprising a first composition comprising a single-stranded guide DNA and a first transfection agent, and a second composition comprising a nucleic acid encoding an Ago protein and a second transfection agent, wherein the Ago protein and the guide DNA form a complex that is capable of specifically recognizing a target locus at a temperature of about 10℃ to about 60℃ (such as about 37℃) , and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for an organism of interest. In some embodiments, the first transfection agent is the same as the second transfection agent. In some embodiments, the first transfection agent is different from the second transfection agent. In some embodiments, the first transfection agent is 2000, a DEAE (diethylaminoethyl) dextran, or HD. In some embodiments, the second transfection agent is 3000.
[0024]
In some embodiments according to any one of the kits described above, the first composition and/or the second composition has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the first composition and/or the second composition has no more than about 2% (v/v) of serum. In some embodiments, the medium is essentially free of serum.
[0025]
Another aspect of the present application provides a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000, DEAE-dextran, or HD) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the method further comprises contacting the cell with a second composition comprising a second nucleic acid and a second transfection agent (e.g., 2000 or 3000) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of the contacting of the cell with the composition comprising the single-stranded DNA. In some embodiments, the cell is at log growth phase at the time of the contacting of the cell with the composition comprising the single-stranded DNA and/or the contacting of the cell with the second composition. In some embodiments, the cell has a confluency of about 30%-80%at the time of the contacting of the cell with the composition comprising the single-stranded DNA and/or the contacting of the cell with the second composition.
[0026]
Yet another aspect of the present application provides a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000, DEAE-dextran, or HD) , wherein the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the method further comprises contacting the cell with a second composition comprising a second nucleic acid and a second transfection agent (e.g., 2000 or 3000) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of the contacting of the cell with the composition comprising the single-stranded DNA. In some embodiments, the cell is at log growth phase at the time of the contacting of the cell with the composition comprising the single-stranded DNA and/or the contacting of the cell with the second composition. In some embodiments, the cell has a confluency of about 30%-80%at the time of the contacting the cell with the composition comprising the single-stranded DNA and/or the contacting of the cell with the second composition.
[0027]
These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]
FIGs. 1A-1C shows images of cells transfected with Alexa-488 labeled ssDNA. In FIG. 1A, cells were cultured in a medium at pH 7.4. Left panel shows a fluorescence microscopy image, and right panel shows a phase contrast image. In FIG. 1B, cells were cultured in a medium at pH 7.6. Left panel shows a fluorescence microscopy image, middle panel shows a phase contrast image, and right panel shows a merged image. In FIG. 1C, cells were cultured in a medium at pH 7.6. Left panel shows a phase contrast image, middle panel shows a fluorescence microscopy image, and right panel shows an image of DAPI-stained cells.
[0029]
FIG. 2A shows percentages of 293T cells transfected with Alexa-488 labeled ssDNA packed with 2000 when cultured in a medium containing 0-10%serum.
[0030]
FIG. 2B shows percentages of HeLa cells transfected with Alexa-488 labeled ssDNA packed with 2000 when cultured in a medium containing 0-10%serum.
[0031]
FIG. 2C shows ssDNA transfection efficiencies using various transfection reagents in a culture medium with or without serum.
[0032]
FIG. 2D shows levels of Alexa488-labeled ssDNA in cells that were supplemented with serum 6 hours after transfection.
[0033]
FIG. 3A shows expression of Flag-NgAgo in cells co-transfected with a plasmid encoding Flag-NgAgo and a ssDNA, in which the plasmid and the ssDNA were co-packed using 2000.
[0034]
FIG. 3B shows GFP expression levels in cells co-transfected with a plasmid encoding GFP and a ssDNA, in which the plasmid and the ssDNA were co-packed or packed separately using 2000.
[0035]
FIG. 4A shows levels of Alex-488 labeled ssDNA in cells after transfection.
[0036]
FIG. 4B shows expression levels of GFP in cells after transfection.
[0037]
FIG. 5A shows a schematic experimental design for inserting an eGFP donor DNA into a DYRK1A locus initiated by NgAgo-gDNA. FIG. 5B shows sequences and binding sites of primers and guide DNA G10. FIG. 5C shows sequencing chromatograms of two PCR amplicons at the DYRK1A and EGFP junction.

DETAILED DESCRIPTION OF THE INVENTION

[0038]
The present invention provides methods, compositions and kits for transfecting a single-stranded DNA into a cell. The methods of ssDNA transfection described herein can be used for gene editing methods that involve DNA-guided nucleases. In some embodiments, methods of modifying a target nucleic acid in a cell by transfecting a single-stranded guide DNA (gDNA) followed by a nucleic acid encoding an Argonaute (Ago) protein. The present invention is based on the discovery that the efficiency of ssDNA transfection is affected by factors including the pH and serum concentration of the cell culture medium. Additionally, separate packing and transfection of ssDNA and a second nucleic acid, such as a plasmid encoding a DNA-guided nuclease (such as an Ago protein) , improves transfection efficiency of the ssDNA.
[0039]
Accordingly, one aspect of the present application provides a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent, wherein the medium has a pH of about 7.2-7.6, and/or wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) .
[0040]
One aspect of the present application provides a method of modifying a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease (such as an Ago protein) into the cell; and subsequently (b) transfecting a single-stranded guide DNA into the cell, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA.
[0041]
Another aspect of the present application provides a method of modifying a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease and a first transfection agent and a second composition comprising a single-stranded guide DNA and a second transfection agent, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA, wherein the medium has a pH of about 7.2-7.6, and/or wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) .
[0042]
Also provided are kits and articles manufacture useful for the methods described herein.
[0043]
I. Definitions
[0044]
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular.
[0045]
As used herein, "Argonaute" and "Ago" are used interchangeable and refer to a naturally occurring or engineered protein that can be guided by a single-stranded oligonucleotide DNA (i.e., guide DNA) to specifically recognize a target nucleic acid comprising a complementary sequence to the guide DNA. Some Ago proteins, also referred herein as “Argonaute nucleases, ” have DNA-guided endonuclease activity, i.e. cleavage of an internal phosphodiester bond in a target nucleic acid. Some Ago proteins do not cleave the target nucleic acid.
[0046]
As used herein, "guide DNA" , "gDNA" , or “DNA guide” are used interchangeably to refer to a single-stranded oligonucleotide DNA that can form a complex with an Argonaute protein of the present application and hybridize to a target nucleic acid. The portion of the target nucleic acid that hybridizes to the guide DNA is referred herein interchangeably as the “target locus” or “target site. ” The complex of an Ago protein bound to a guide DNA is referred herein as “Ago-gDNA” or “Ago-G. ”
[0047]
As used herein, "donor DNA" refers to a polynucleotide that can be integrated into the site of a double-strand break induced by an Ago-gDNA complex.
[0048]
The terms “nucleic acid, ” “polynucleotide, ” and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof. “Oligonucleotide” and “oligo” are used interchangeably to refer to a short polynucleotide, having no more than about 50 nucleotides.
[0049]
"Complementarity" refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, 10 out of 10, being about 50%, 60%, 70%, 80%, 90%, and 100%complementary respectively) . "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least about any one of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%over a region of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
[0050]
“Mismatch” refers to a nucleotide in a first nucleic acid that does not form a traditional Watson-Crick basepair with a corresponding nucleotide in a second nucleic acid.
[0051]
As used herein, “target” or “targeting” refers to specific binding of a gDNA or an Ago-gDNA complex to a nucleic acid. “Specific binding” refers to hybridization under stringent conditions.
[0052]
As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993) , Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay" , Elsevier, N, Y.
[0053]
"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
[0054]
“Percentage (%) sequence identity” with respect to a peptide, polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. “Percentage (%) sequence homology” with respect to a peptide, polypeptide or protein sequence is the percentage of amino acid residues in a candidate sequence that are identical or conservative substitutions to amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
[0055]
The terms "polypeptide" , and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may he linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. A protein may have one or more polypeptides. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
[0056]
The terms "transfect, " "transform, " "transduce, " and “deliver” are used interchangeably herein to refer to a process by which exogenous molecules (such as nucleic acids, proteins, or complexes thereof) are transferred or introduced into a cell.
[0057]
The term “cell” includes the primary subject cell and its progeny. The terms “host cell” refers to cells into which exogenous nucleic acids or protein complexes (such as Ago-gDNA complex) have been introduced, including the progeny of such cells. Cells and host cells include “transformants” and “transfected cells, ” which include the primary transfected cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transfected cell are included herein.
[0058]
As used herein, the term "isolated" can refer to a nucleic acid or polypeptide that, by the hand of a human, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form and/or can exist in a non-native environment such as, for example, in a transgenic cell.
[0059]
It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of” embodiments.
[0060]
Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X" .
[0061]
As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter.
[0062]
The term “about X-Y” used herein has the same meaning as “about X to about Y. ”
[0063]
As used herein, the singular forms "a, " "or, " and "the" include plural referents unless the context clearly dictates otherwise.
[0064]
II. Methods of transfecting a single-stranded DNA
[0065]
The present application provides methods of transfecting a single-stranded DNA into a cell. Various conditions affect the transfection efficiency of ssDNA, including, for example, pH and serum concentration of medium containing the cell, and other nucleic acid (s) that are co-transfected into the cell.
[0066]
In some embodiments, there is provided a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6.
[0067]
In some embodiments, there is provided a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000) , wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) . In some embodiments, the cell is first cultured in a medium having serum at a concentration of about 5%(v/v) or more, such as about 10% (v/v) , and then transferred to a medium having no more than about 2% (v/v) of serum (such as essentially free of serum) prior to the contacting. In some embodiments, the cell is further cultured in a medium having serum at a concentration of about 5%(v/v) or more, such as about 10% (v/v) , after at least about 2 hours (such as at least about any one of 3, 4, 5, 6, or more hours) of the contacting.
[0068]
In some embodiments, there is provided a method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6, and wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) . In some embodiments, the cell is first cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , and then transferred to a medium having serum at a concentration of no more than about 2% (v/v) of serum (such as essentially free of serum) prior to the contacting. In some embodiments, the cell is further cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , after at least about 2 hours (such as at least about any one of 3, 4, 5, 6, or more hours) of the contacting.
[0069]
In some embodiments, there is provided a method of transfecting a single-stranded DNA and a second nucleic acid into a cell, comprising contacting the cell in a medium with a first composition comprising the single-stranded DNA and a first transfection agent (e.g., 2000) and a second composition comprising the second nucleic acid and a second transfection agent (e.g., 3000 or 2000) . In some embodiments, the first composition and the second composition are not admixed prior to the contacting. In some embodiments, the first transfection agent is the same as the second transfection agent. In some embodiments, the first transfection agent is different from the second transfection agent. In some embodiments, the medium has a pH of about 7.2-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) . In some embodiments, the cell is first cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , and then transferred to a medium having serum at a concentration of no more than about 2% (v/v) (such as essentially free of serum) prior to the contacting. In some embodiments, the cell is further cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , after at least about 2 hours (such as at least about any one of 3, 4, 5, 6, or more hours) of the contacting.
[0070]
In some embodiments, there is provided a method of transfecting a single-stranded DNA and a second nucleic acid into a cell, comprising: (a) contacting the cell in a medium with a first composition comprising the single-stranded DNA and a first transfection agent (e.g., 2000) , and subsequently (b) contacting the cell in the medium with a second composition comprising the second nucleic acid and a second transfection agent (e.g., 3000 or 2000) . In some embodiments, step (b) is carried out after about 2-48 hours of step (a) . In some embodiments, the first transfection agent is the same as the second transfection agent. In some embodiments, the first transfection agent is different from the second transfection agent. In some embodiments, the medium has a pH of about 7.2-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) . In some embodiments, the cell is first cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , and then transferred to a medium having serum at a concentration of no more than about 2% (v/v) (such as essentially free of serum) prior to step (a) . In some embodiments, the cell is further cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , after at least about 2 hours (such as at least about any one of 3, 4, 5, 6, or more hours) of step (a) .
[0071]
In some embodiments, there is provided a method of transfecting a single-stranded DNA and a second nucleic acid into a cell, comprising: (a) contacting the cell in a medium with a first composition comprising the second nucleic acid and a first transfection agent (e.g., 3000 or 2000) , and subsequently (b) contacting the cell in the medium with a second composition comprising the single-stranded DNA and a second transfection agent (e.g., 2000) . In some embodiments, step (b) is carried out after about 2-48 hours of step (a) . In some embodiments, the first transfection agent is the same as the second transfection agent. In some embodiments, the first transfection agent is different from the second transfection agent. In some embodiments, the medium has a pH of about 7.2-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) . In some embodiments, the cell is first cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10% (v/v) , and then transferred to a medium having serum at a concentration of no more than about 2% (v/v) (such as essentially free of serum) prior to step (b) . In some embodiments, the cell is further cultured in a medium having serum at a concentration of about 5% (v/v) or more, such as about 10%(v/v) , after at least about 2 hours (such as at least about any one of 3, 4, 5, 6, or more hours) of step (b) .
[0072]
In some embodiments, non-alkaline medium increases the transfection efficiency of single-stranded DNA. In some embodiments, the cell is transfected with the single-stranded DNA in a medium having a pH of about 7.2-7.6. In some embodiments, the medium has a pH of about any one of 7.2-7.3, 7.3-7.4, 7.4-7.5, 7.5-7.6, 7.2-7.4, 7.4-7.6, or 7.3-7.5. In some embodiments, the medium has a pH no more than about any one of 7.6, 7.5, 7.4, or 7.3. In some embodiments, the medium has a pH of about any one of 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, or 7.6.
[0073]
In some embodiments, low serum level in the medium increases the transfection efficiency of single-stranded DNA. In some embodiments, the medium has a serum level of no more than about any one of 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%or less. In some embodiments, the medium is essentially free of serum. In some embodiments, the serum level is measured as a volume/volume (v/v) percentage with respect to the total transfection mixture. In some embodiments, the serum level is measured as a weight/volume (w/v) percentage with respect to the total transfection mixture. In some embodiments, the serum level is measured as a weight/weight (w/w) percentage with respect to the total transfection mixture. In some embodiments, the serum is fetal bovine serum (i.e., FBS) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more, such as more than about any one of 8%, 10%, 15%, 20%, 30%, 40%or more. In some embodiments, the medium is supplemented with serum after at least about 2 hours (e.g., at least about any one of 3, 4, 5, 6, 7, 8 or more hours) of the contacting of the cell with the single-stranded DNA. In some embodiments, the medium is supplemented with serum to a final concentration of about 10%(v/v) after about 6 hours of the contacting of the cell with the single-stranded DNA.
[0074]
In some embodiments, the cell is at log growth phase at the time of the transfection of the ssDNA. In some embodiments, the cell has a confluency of about 30%-80% (such as about any one of 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 30%-50%, 40%-60%, 60%-80%, or 40%-60%) at the time of the contacting of the cell with the single-stranded DNA. In some embodiments, the cell is at no more than about any one of 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80%confluency at the time of the contacting of the cell with the single-stranded DNA.
[0075]
In some embodiments, a composition comprising the single-stranded DNA and a transfection agent is obtained. In some embodiments, the single-stranded DNA is “packed” with a transfection agent by mixing the transfection agent with the single-stranded DNA. Many transfection agents for nucleic acid may be suitable for transfecting single-stranded DNA, including, but not limited to, dendrimers, liposomes, and cationic polymers (e.g., DEAE-dextran or polyethylenimine) . In some embodiments, the transfection agent is 2000. In some embodiments, the transfection agent is DEAE-dextran. In some embodiments, the transfection agent is HD. In some embodiments, the transfection agent is not 3000.
[0076]
The single-stranded DNA can be of any suitable length and comprise any suitable sequences. In some embodiments, the single-stranded DNA is at least about any one of 10 nucleotides ( “nt” ) , 20 nt, 50 nt, 100 nt, 200 nt, 500 nt, 1000 nt, or longer. In some embodiments, the single-stranded DNA is about 10 to about 50 nt, such as any one of about 10 nt to about 20 nt, about 20 nt to about 30 nut, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 15 nt to about 30 nt, about 20 nt to about 40 nt, about 15 nt to about 25 nt, or about 20 nt to about 35 nt. In some embodiments, the single-stranded DNA has about any one of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the single-stranded DNA has one or more modified nucleotide, including nucleobase modification and/or backbone modification (e.g., phosphorothioate modification) . In some embodiments, the single-stranded DNA does not have modifications to the nucleobase or backbone. In some embodiments, the single-stranded DNA has a 5’ phosphate group. In some embodiments, the single-stranded DNA is a guide DNA for an Argonaute protein.
[0077]
In some embodiments, the method comprises transfecting a second nucleic acid into the cell. In some embodiments, the second nucleic acid is a second single-stranded DNA. In some embodiments, the method comprises transfecting a plurality (such as at least about any one of 2, 3, 4, 5, 10 or more different species) of single-stranded DNA into the cell. In some embodiments, the second nucleic acid is a double-stranded DNA. In some embodiments, the second nucleic acid is a linear nucleic acid. In some embodiments, the second nucleic acid is a plasmid. In some embodiments, the second nucleic acid is a linearized plasmid. In some embodiments, the second nucleic acid is a nucleic acid (such as a vector, e.g., a plasmid or a viral vector) encoding an Argonaute protein. The second nucleic acid can be of any suitable length and sequence, including, but not limited to, at least any one of 500bp, 1kb, 2kb, 5kb, 10 kb or longer. In some embodiments, the molecular ratio between the second nucleic acid and the single-stranded DNA is no more than about any one of 100: 1, 50: 1, 10: 1, 5: 1, 2: 1, 1: 1, 1: 5, 1: 10 or less. In some embodiments, the method comprises transfecting a plurality (such as any one of 2, 3, 4, 5, or more different species) of nucleic acids into the cell besides the ssDNA.
[0078]
The second nucleic acid and any other additional nucleic acids may be transfected into the cell using any known methods and known transfection agents in the art. Exemplary intracellular transfection methods, include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine) ; non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, the second nucleic acid is transfected using the same transfection agent as the ssDNA. In some embodiments, the second nucleic acid is transfected using a different transfection agent as the ssDNA. In some embodiments, the transfection agent is 2000. In some embodiments, the transfection agent is DEAE-dextran. In some embodiments, the transfection agent is HD. In some embodiments, the transfection agent is 3000.
[0079]
In some embodiments, the second nucleic acid and any other additional nucleic acids are transfected into the cell simultaneously as the single-stranded DNA. In some embodiments, the second nucleic acid and any other additional nucleic acids are transfected into the cell before the transfection of the single-stranded DNA. In some embodiments, the second nucleic acid and any other additional nucleic acids are transfected into the cell after the transfection of the single-stranded DNA. In some embodiments, the interval between the transfection of the single- stranded nucleic acid and the transfection of the second nucleic acid and any other additional nucleic acids is at least about any one of 2, 3, 4, 5, 6, 8, 10, 12, 18, 24, 30, 36, 42, or 48 hours. In some embodiments, the interval between the transfection of the single-stranded nucleic acid and the transfection of the second nucleic acid and any other additional nucleic acids is about any one of 2-48 hours, 2-12 hours, 12-24 hours, 24-36 hours, 36-48 hours, 2-24 hours, 6-12 hours, 12-36 hours, or 6-18 hours. In some embodiments, the number of transfections is minimized to avoid toxicity of liposomes to cells.
[0080]
In some embodiments, wherein the second nucleic acid is transfected simultaneously as the ssDNA, the second nucleic acid and the ssDNA are separately packed with the transfection agent. In some embodiments, the method comprises contacting the cell in a medium a first composition comprising an ssDNA and a first transfection agent and a second composition comprising the second nucleic acid and a second transfection agent.
[0081]
Further provided are transfected cells produced by the methods described herein, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
[0082]
III. Methods of modifying a target nucleic acid in a cell
[0083]
The present application provides methods of modifying a target nucleic acid in a cell comprising co-transfection of separately packed single-stranded guide DNA (gDNA) and a nucleic acid (such as a vector) encoding a DNA-guided nuclease, or transfection of a single-stranded gDNA into the cell followed by transfection of a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) . In some embodiments, the method further comprises transfecting a donor DNA into the cell. In some embodiments, the donor DNA is co-transfected with the gDNA, wherein the donor DNA and the gDNA are separately packed in a transfection agent. In some embodiments, the donor DNA is transfected after the transfection of the gDNA. Any of the transfection methods described in Section II can be used for transfecting the single-stranded gDNA. The methods described herein can be used for intracellular gene editing. The target nucleic acid can be an endogenous nucleic acid (such as genomic DNA or RNA) in the cell, or an exogenous nucleic acid, such as a viral nucleic acid, in the cell. The methods described herein are compatible with a variety of cells, including both prokaryotic cells and eukaryotic cells.
[0084]
Thus, in some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) into the cell; and subsequently (b) transfecting a single- stranded guide DNA into the cell, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0085]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) into the cell; and subsequently (b) contacting the cell in a medium with a composition comprising a single-stranded guide DNA and a transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) , wherein the a DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0086]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) into the cell; and subsequently (b) contacting the cell in a medium with a composition comprising a single-stranded guide DNA and a transfection agent (e.g., 2000) , wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) , wherein the a DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0087]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0088]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0089]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) , wherein the medium has no more than about 2% (v/v) of serum (such as essentially free of serum) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0090]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease (such as an Argonaute protein) and a first transfection agent (e.g., 3000) and a second composition comprising a single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of the contacting. In some embodiments, the cell is at log growth phase at the time of the contacting. In some embodiments, the cell has a confluency of about 30%-80%at the time of the contacting.
[0091]
As used herein, “DNA-guided nuclease” refers to any nuclease that binds to a single-stranded DNA and recognizes a target nucleic acid based on the sequence of the single-stranded DNA. In some embodiments, the DNA-guided cleaves the target locus. In some embodiments, wherein the target locus is a double-stranded DNA, the DNA-guided nuclease protein induces a double-strand break in the target locus. In some embodiments, the DNA-guided nuclease does not cleave the target locus. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%.
[0092]
In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the nucleic acid encoding the DNA-guided nuclease is codon-optimized for the organism from which the cell is derived. In some embodiments, the DNA-guided nuclease comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is a genomic DNA. In some embodiments, the target nucleic acid is a RNA, such as an mRNA. In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is integrated in the genome of the cell. In some embodiments, the target nucleic acid is not integrated in the genome of the cell. In some embodiments, the target nucleic acid is a viral DNA. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as plant, fungal, yeast, or mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is derived from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the cell is an immune cell.
[0093]
In some embodiments, the DNA-guided nuclease is an Argonaute protein. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42.
[0094]
Thus, in some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding an Argonaute protein into the cell; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell, wherein the Ago protein and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid and induces a double-strand break (DSB) in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0095]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding an Argonaute protein and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a 5’ phosphorylated single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) , wherein the Ago protein and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid and induces a double-strand break (DSB) in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5%(v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0096]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding an Argonaute protein and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a 5’ phosphorylated single-stranded guide DNA and a second transfection agent (e.g., 2000) , has no more than about 2% (v/v) of serum (such as essentially free of serum) , wherein the Ago protein and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid and induces a double-strand break (DSB) in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0097]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: (a) contacting the cell in a medium with a first composition comprising a nucleic acid encoding an Argonaute protein and a first transfection agent (e.g., 3000) ; and subsequently (b) contacting the cell in the medium with a second composition comprising a 5’ phosphorylated single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the medium has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) , wherein has no more than about 2% (v/v) of serum, such as essentially free of serum, wherein the Ago protein and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid and induces a double-strand break (DSB) in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 after step (a) . In some embodiments, the medium is supplemented with serum to a final concentration of about 5%(v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0098]
In some embodiments, there is provided a method of modifying of a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding an Argonaute protein and a first transfection agent (e.g., 3000) and a second composition comprising a 5’ phosphorylated single-stranded guide DNA and a second transfection agent (e.g., 2000) , wherein the Ago protein and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid and induces a double-strand break (DSB) in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) .
[0099]
In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to SEQ ID NO: 1. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the organism from which the cell is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is a genomic DNA. In some embodiments, the target nucleic acid is a RNA, such as an mRNA. In some embodiments, the target nucleic acid is exogenous to the cell. In some embodiments, the target nucleic acid is integrated in the genome of the cell. In some embodiments, the target nucleic acid is not integrated in the genome of the cell. In some embodiments, the target nucleic acid is a viral DNA. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell, such as plant, fungal, yeast, or mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is derived from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the cell is an immune cell.
[0100]
The nucleic acid encoding the Ago protein and the single-stranded guide DNA can be transfected into the cell using any suitable methods known in the art, or any one of the methods of transfection described herein. In some embodiments, the nucleic acid encoding the Ago protein and the single-stranded guide DNA are transfected into the cell simultaneously, wherein the nucleic acid encoding the Ago protein and the single-stranded guide DNA are packed with the transfection agent separately.
[0101]
In some embodiments, the peak expression level of the Ago protein in the cell occurs about 24 hours to 36 hours after transfection. In some embodiments, the peak level of single-stranded guide DNA in the cell occurs about 6 hours after transfection. In some embodiments, to optimize the formation of Ago-gDNA complex, the single-stranded guide DNA is transfected into the cell about 2-48 hours, such as about any one of 2-12 hours, 12-24 hours, 24-36 hours, 36-48 hours, 2-24 hours, 6-12 hours, 12-36 hours, or 6-18 hours after the transfection of the nucleic acid encoding the Ago protein.
[0102]
In some embodiments, the gDNA is transfected into the cell for more than one time. In some embodiments, a first batch of the gDNA is transfected into the cell about 2-48 hours (such as about 6-12 hours) after the transfection of the nucleic acid encoding the Ago protein, and subsequently one or more additional batches of the gDNA were transfected into the cell after at least about any one of 2, 4, 6, 8, 10, 12, 16, 20, 24, or more hours after the first transfection of the single-stranded gDNA.
[0103]
The Ago proteins described herein can modify a target nucleic acid in a cell in a variety of ways. In some embodiments, the method induces a site-specific cleavage in the target nucleic acid. In some embodiments, the method cleaves a genomic DNA in a bacterial cell. In some embodiments, the method cleaves a viral nucleic acid in a cell. In some embodiments, the method alters (such as increase or decrease) the expression level of the target nucleic acid in the cell. In some embodiments, the method reduces or silences the expression level of the target nucleic acid in the cell. In some embodiments, the method uses one or more endogenous DNA repair pathways, such as Non-homologous end joining (NHEJ) or Homology directed recombination (HDR) , in the cell to repair the double-strand break induced in the target locus by the Ago protein, thereby introducing mutations or exogenous sequences at the target locus. In some embodiments, the method introduces a mutation at the target locus. Exemplary mutations include, but are not limited to, insertions, deletions, substitutions, and frameshifts. In some embodiments, the method inserts a donor DNA at the target locus. In some embodiments, the insertion of the donor DNA results in introduction of a selection marker or a reporter protein to the cell. In some embodiments, the insertion of the donor DNA results in knock-in of a gene. In some embodiments, the insertion of the donor DNA results in a knockout mutation. In some embodiments, the insertion of the donor DNA results in a substitution mutation, such as a single nucleotide substitution. In some embodiments, the method induces a phenotypic change to the cell.
[0104]
Thus, in some embodiments, there is provided a method of site-specific cleavage of a target nucleic acid (such as a viral nucleic acid) in a cell comprising: (a) transfecting a nucleic acid encoding an Argonaute protein (such as NgAgo) into the cell; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell, wherein the Ago protein and the guide DNA form a complex that specifically cleaves a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, wherein the target locus is a double-stranded DNA, the Ago protein induces a double-strand break in the target locus. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the cell is transfected with the guide DNA for at least two (such as about any one of 2, 3, 4, 5, 6, or more) times. In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the Ago protein is at least about 100: 1. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the organism from which the cell is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid (such as at least about 95%supercoiled) . In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the method induces a phenotypic change in the cell.
[0105]
In some embodiments, there is provided a method of inhibiting growth of a target cell (such as bacterial cell) comprising transfecting: (a) transfecting a nucleic acid encoding an Argonaute protein (such as NgAgo) into the cell; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell, wherein the Ago protein and the guide DNAs form complexes that specifically recognize and cleaves one or more target loci in the genomic DNA, and wherein the target loci comprise complementary sequences to the one or more guide DNAs. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the Ago protein is derived from Natronobacterium gregoryi. In some embodiments, the vector is linearized prior to the transfection. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the target cell.
[0106]
In some embodiments, there is provided a method of altering (such as decreasing) expression of a target nucleic acid (such as a gene) in a cell comprising : (a) transfecting a nucleic acid encoding an Argonaute protein (such as NgAgo) into the cell; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell, wherein the Ago protein and the guide DNA form a complex that specifically cleaves a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, the complex cleaves the target locus. In some embodiments, wherein the target locus is a double-stranded DNA, the Ago protein induces a double-strand break in the target locus. In some embodiments, the complex does not cleave the target locus. In some embodiments, the guide DNA does not dissociate from the Ago protein at a temperature lower than about 50℃. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the cell is transfected with the guide DNA for at least two (such as about any one of 2, 3, 4, 5, 6, or more) times. In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the Ago protein is at least about 100: 1. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the organism from which the cell is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid (such as at least about 95%supercoiled) . In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the target locus is a disease-associated locus. In some embodiments, the method induces a phenotypic change in the cell.
[0107]
In some embodiments, there is provided a method of introducing a mutation (such as indel or frameshift mutation) in a target nucleic acid (such as genomic DNA) in a cell comprising: (a) transfecting a nucleic acid encoding an Argonaute protein (such as NgAgo) into the cell; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell, wherein the Ago protein and the guide DNA form a complex that specifically cleaves a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the cell is transfected with the guide DNA for at least two (such as about any one of 2, 3, 4, 5, 6, or more) times. In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the Ago protein is at least about 100: 1. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the organism from which the cell is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid (such as at least about 95%supercoiled) . In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the target locus is a disease-associated locus. In some embodiments, the method induces a phenotypic change in the cell.
[0108]
In some embodiments, the method further comprises contacting the target nucleic acid with a donor DNA comprising a sequence homologous to the sequence of the target locus under a condition that allows integration of the donor DNA at the target locus. The donor DNA can be delivered into the cell using any known methods in the art. In some embodiments, the donor DNA is delivered into the cell simultaneously as the nucleic acid encoding the Ago protein and/or the guide DNA, or sequentially (e.g., after) the nucleic acid encoding the Ago protein and/or the guide DNA. In some embodiments, the donor DNA is transfected into the cell about 2-15 hours, such as about any one of 4-12, 6-10, or 6-12 hours after the transfection of the single-stranded guide DNA. In some embodiments, the donor DNA and the guide DNA are transfected into the cell simultaneously, and wherein the donor DNA and the guide DNA are separately packed with a transfection agent (such as 2000) .
[0109]
In some embodiments, there is provided a method of inserting a donor DNA in a target nucleic acid (such as genomic DNA) in a cell comprising: (a) transfecting a nucleic acid encoding an Argonaute protein (such as NgAgo) into the cell; subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the cell; and (c) transfecting a donor DNA into the cell, wherein the Ago protein and the guide DNA form a complex that specifically cleaves a target locus in the target nucleic acid, and induces a double-strand break in the target locus, wherein the donor DNA is integrated at the DSB in the target locus, wherein the target locus is a double-stranded DNA comprising a sequence that is complementary to the sequence of the guide DNA, and wherein the donor DNA comprises a sequence homologous to the sequence of the target locus. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the cell is transfected with the nucleic acid encoding the Argonaute protein, the single-stranded guide DNA, and/or the donor DNA in a medium. In some embodiments, the medium is at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5%(v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, the donor DNA and the guide DNA are transfected into the cell separately. In some embodiments, the donor DNA is transfected into the cell at least about 6 hours after the transfection of the guide DNA. In some embodiments, the donor DNA and the guide DNA are transfected into the cell simultaneously, and wherein the donor DNA and the guide DNA are separately packed with a transfection agent. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the cell is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the cell is transfected with the guide DNA for at least two (such as about any one of 2, 3, 4, 5, 6, or more) times. In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the Ago protein is at least about 100: 1. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the organism from which the cell is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the cell is treated with one or more antibiotics. In some embodiments, the cell is free from contamination by (other) non-viral microorganisms. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid (such as at least about 95%supercoiled) . In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the target locus is a disease-associated locus. In some embodiments, the method induces a phenotypic change in the cell. In some embodiments, wherein the donor DNA comprises an exogenous sequence (such as an exogenous gene) , the method introduces a knock-in of the exogenous sequence at the target locus. In some embodiments, wherein the donor DNA comprises a substitution mutation (such as a single nucleotide substitution) , the method introduces the substitution mutation at the target locus. In some embodiments, the method introduces a knockout mutation at the target locus.
[0110]
The nucleic acid encoding the Ago protein, the guide DNA, and the donor DNA can be transfected into the cell using the same method or different methods. Suitable methods can be chosen by a skilled person in the art among a variety of known methods. Suitable amounts of the nucleic acid encoding the Ago protein, the guide DNA, and the donor DNA can be chosen and adjusted depending on the method (s) of transfection, order of transfection, ad nature of the nucleic acids transfected. In some embodiments, at least about any one of 10 ng, 50 ng, 100 ng, 200 ng, 300 ng, 500 ng, 750 ng, 1mg or more of each nucleic acid encoding the Ago protein, the guide DNA and optionally the donor DNA is transfected into the cell. In some embodiments, about 100ng to 500 ng of each nucleic acid encoding the Ago protein, the guide DNA and optionally the donor DNA is transfected into the cell. In some embodiments, wherein a plasmid encoding the Ago protein and the guide DNA are transfected simultaneously into the cell, the weight ratio between the guide DNA and the plasmid is at least about 1: 1, 1: 2, 1: 3, 1: 4, or 1: 5. In some embodiments, the molar ratio between the guide DNA and the nucleic acid (such as vector) encoding the Ago protein transfected into the cell is at least about any one of 10: 1, 20: 1, 50: 1, 100: 1, 150: 1, 200: 1, 300: 1, 500: 1, 750: 1, 1000: 1 or more. In some embodiments, the molar ratio between the guide DNA and the donor DNA transfected into the cell is at least about any one of 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1 or more. In some embodiments, the molar ratio between the donor DNA and the nucleic acid (such as vector) encoding the Ago protein transfected into the cell is at least about any one of 10: 1, 20: 1, 50: 1, 100: 1, 150: 1, 200: 1, 300: 1, 500: 1, 750: 1, 1000: 1 or more.
[0111]
Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are sold commercially (e.g., TRANSFECTAMINE TM and ) . In some embodiments, 2000 is used to transfect the gDNA and/or the donor DNA. In some embodiments, 3000 is used to transfect the nucleic acid encoding Ago (such as vector) .
[0112]
Conventional viral based systems for nucleic acid delivery include retroviral, lenti virus, adenoviral, adeno-associated and herpes simplex virus vectors. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus methods, often resulting in long term expression of the inserted transgene. Additionally, high transfection efficiencies have been observed in many different cell types. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transfect or infect non-dividing cells and typically produce high viral titers. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the nucleic acids into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia vims (MuLV) , gibbon ape leukemia virus (GaLV) , Simian Immuno deficiency vims (SIV) , human immuno deficiency vims (HIV) , and combinations thereof. In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transfection efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
[0113]
Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293T cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide (s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
[0114]
In some embodiments, the method comprises one or more steps assessing the cell after the modification. In some embodiments, the method comprises assessing the cell for a phenotypic change. In some embodiments, wherein the donor DNA comprises a selection marker, such as a reporter protein, the method comprises assessing the cell for expression of the selection marker. In some embodiments, the method comprises sequencing the target nucleic acid. In some embodiments, the method comprises modifying the target nucleic acid in a plurality of cells, and selecting a cell having a modified target nucleic acid based on one or more of the following: (1) a phenotypic change to the cell; (2) expression of a selection marker, such as a reporter protein, wherein the donor DNA comprises the selection marker; and/or (3) sequence of the modified target nucleic acid in the cell.
[0115]
Thus, in some embodiments, there is provided a method comprising modifying a target nucleic acid in a plurality of cells, comprising: (a) transfecting a nucleic acid encoding an Argonaute protein into the plurality of cells; and subsequently (b) transfecting a 5’ phosphorylated single-stranded guide DNA into the plurality of cells, wherein the Ago protein and the guide DNA form a complex that specifically cleaves a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA; and (c) selecting a cell having a modified target nucleic acid based on one or more of the following: (1) a phenotypic change to the cell; (2) expression of a selection marker (such as a reporter protein) , wherein the method further comprises contacting the target nucleic acid with a donor DNA comprising a sequence homologous to the sequence of the target locus under a condition that allows integration of the donor DNA at the target locus, and wherein the donor DNA comprises the selection marker; and/or (3) sequence of the modified target nucleic acid in the cell. In some embodiments, step (b) is carried out about 2-48 hours, such as about 2-12 hours or about 6-18 hours after step (a) . In some embodiments, the plurality of cells is transfected with the nucleic acid encoding the Argonaute protein and/or the single-stranded guide DNA in a medium at pH 7.4-7.6. In some embodiments, the medium has no more than about 2% (v/v) of serum, such as essentially free of serum. In some embodiments, the medium is supplemented with serum to a final concentration of about 5% (v/v) or more (e.g., about 10%or more) after at least about 2 hours (e.g., at least about 6 hours) of step (b) . In some embodiments, the cell is at log growth phase in step (a) and/or step (b) . In some embodiments, the cell has a confluency of about 30%-80%in step (a) and/or step (b) . In some embodiments, the complex cleaves the target locus. In some embodiments, the donor DNA and the guide DNA are transfected into the cell separately. In some embodiments, the donor DNA is transfected into the cell at least about 6 hours after the transfection of the guide DNA. In some embodiments, the donor DNA and the guide DNA are transfected into the cell simultaneously, and wherein the donor DNA and the guide DNA are separately packed with a transfection agent. In some embodiments, wherein the target locus is a double-stranded DNA, the Ago protein induces a double-strand break in the target locus. In some embodiments, the complex does not cleave the target locus. In some embodiments, the guide DNA does not dissociate from the Ago protein at a temperature lower than about 50℃. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site. In some embodiments, the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site. In some embodiments, the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80%sequence homology (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) to a sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the guide DNA is about 10 to about 50 (such as about 20 to about 30) nucleotides long. In some embodiments, the plurality of cells is supplemented with a divalent metal ion (such as at least about 0.1 mM) . In some embodiments, the molar ratio of the guide DNA to the nucleic acid encoding the Ago protein is at least about 100: 1. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for the species from which the plurality of cells is derived. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) . In some embodiments, the plurality of cells is treated with one or more antibiotics. In some embodiments, the plurality of cells is free from contamination by (other) non-viral microorganisms. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid (such as at least about 95%supercoiled) . In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the modifying comprises introducing a mutation (such as an indel or frameshift mutation) at the target locus. In some embodiments, the modifying comprises altering expression of the target nucleic acid. In some embodiments, the modifying comprises introducing a knockout mutation at the target locus. In some embodiments, the modifying comprises inducing a phenotypic change to the cell. In some embodiments, the modifying comprises introducing a knock-out mutation, a knock-in of an exogenous sequence, or a substitution (such as single-nucleotide substitution) mutation at the target locus.
[0116]
Further provided are transfected cells produced by the methods described herein, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
[0117]
A. Argonautes
[0118]
The methods described herein are based on Argonautes that are capable of using a single-stranded guide DNA to specifically recognize a target nucleic acid comprising a perfectly or substantially complementary sequence to the guide DNA at a temperature of about 10℃ to about 60℃, such as any one of about 10℃ to about 20℃, about 20℃ to about 30℃, about 30℃ to about 40℃, about 40℃ to about 50℃, about 50℃ to about 60℃, about 20℃ to about 40℃, about 10℃ to about 50℃, or about 15℃ to about 45℃. In some embodiments, the Argonaute is active at about any one of 10℃, 15℃, 20℃, 25℃, 30℃, 32℃, 34℃, 36℃, 37℃, 38℃, or 40℃. In some embodiments, the activity of the Argonaute is reduced by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%80%, 90%or more at least about 50℃.
[0119]
The activity of the Argonaute includes specific binding and cleavage of a target locus. In some embodiments, the Argonaute is an endonuclease that cleaves the target nucleic acid at the target locus that hybridizes to the guide DNA. In some embodiments, the Ago-gDNA complex cleaves one strand of a double-stranded target DNA. In some embodiments, the Ago-gDNA complex cleaves both strands of a double-stranded target DNA. In some embodiments, the Ago-gDNA complex cleaves RNA, such as an mRNA. In some embodiments, the Argonaute does not have nuclease activity, i.e., the Argonaute forms a complex with the gDNA to specifically recognize (i.e., specifically bind) to the target locus without cleaving the target locus. Nuclease activity of the Ago-gDNA complex can be determined using known methods in the art, such as in vitro plasmid cleavage assay, T7E1 assay, and sequencing. See, for example, experimental protocols in the Example section. Target nucleic acid binding activity of the Ago-gDNA complex can be determined using known methods in the art, such as Electrophoretic Mobility Shift Assay (EMSA) .
[0120]
The Argonautes suitable for the methods in the present application may further have one or more of the following characteristics: (1) the guide DNA is phosphorylated at the 5’ end; (2) once forming a complex, the guide DNA does not dissociate from the Ago protein at a temperature lower than about 50℃; (3) the Ago nuclease requires only a single gDNA to induce a double-strand break in a double-stranded target DNA; (4) the Ago-gDNA complex can specifically recognize and/or cleave a target locus having no more than about 3 mismatches to the sequence of the gDNA; (5) the Ago-gDNA complex can specifically recognize and/or cleavage a target locus having a GC content of at least about 60%; (6) the Ago protein comprises at least 2 of the 3 conservative amino acids in the KQK motif of the 5’ -phosphate binding site; and (7) the Ago protein comprises at least 2 of the 3 conservative amino acids in the DDE motif of the nuclease active site.
[0121]
The Argonaute proteins having the properties described above may be derived from naturally-occurring Argonautes from a variety of organisms. In some embodiments, the Argonaute is derived from a prokaryotic species, such as an archaea or a bacterium. In some embodiments, the Ago is derived from a non-thermophilic bacterium. In some embodiments, the Ago is derived from a mesophilic bacterium. In some embodiments, the Ago is not derived from a thermophilic bacterium. In some embodiments, the Ago is derived from an algal species. In some embodiments, the Ago is derived from Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium, or Pedobacter heparinus. In some embodiments, the Ago is derived from Synechococcus sp., Lyngbya sp., Microcystis sp, Halogeometricum borinquense, Natrinema pellirubrum, Natronobacterium gregoryi, Natronorubrum tibetense, Thermosynechococcus elongatus, Halogeometricum pallidum, Pedobacter heparinus, Rhodobacterales bacterium, Mesorhizobium loti, Haloarcula marismortui, Burkholderia graminis, Burkholderia ambifaria, Natrialba asiatica, or Microcystis aeruginosa. In some embodiments, the Ago is derived from Natronobacterium gregoryi, such as N. gregoryi SP2. In some embodiments, the Ago is derived from Microcystis aeruginosa, such as Microcystis aeruginosa NIES 843. In some embodiments, the Ago is derived from Microcystis sp., such as Microcystis sp. 7806. In some embodiments, the Ago is derived from Pedobacter heparinus, such as Pedobacter heparinus DSM 2366. In some embodiments, the Ago is not derived from Thermus thermophilus or Rhodobacter sphaeroides. In some embodiments, the Ago is not derived from Synechococcus elongatus.
[0122]
In some embodiments, the Argonaute is a type I prokaryotic Argonaute. In some embodiments, the type I prokaryotic Argonaute carries a DNA-targeting guide DNA. In some embodiments, the DNA nucleic acid-targeting nucleic acid targets one strand of a double stranded DNA (dsDNA) to produce a nick or a break of the dsDNA. In some embodiments, the nick or break triggers host DNA repair. In some embodiments, the host DNA repair is non-homologous end joining (NHEJ) or homology directed recombination (HDR) . In some embodiments, the type I prokaryotic Argonaute is a long type I prokaryotic Argonaute. In some embodiments, the long type I prokaryotic Argonaute possesses an N-PAZ-MID-PIWI domain architecture. In some embodiments the long type I prokaryotic Argonaute possesses a catalytically active PIWI domain. In some embodiments, the long type I prokaryotic Argonaute possesses a catalytic tetrad comprising an aspartate-glutamate-aspartate-aspartate/histidine (DEDX) motif. In some embodiments, the catalytic tetrad binds one or more divalent metal ions, such as Mg 2+, or Mn 2+. In some embodiments, the type I prokaryotic Argonaute anchors the 5'phosphate end of a guide DNA.
[0123]
The Argonaute protein may comprise one or more domains. The Argonaute protein may comprise a domain selected from a PAZ domain, a MID domain, and a PIWI domain or any combination thereof. The Argonaute protein may comprise a domain architecture of N-PAZ-MID-PIWI-C. The PAZ domain may comprise an oligonucleotide-binding fold to secure a 3'end of a guide DNA. Release of the 3 '-end of the guide DNA from the PAZ domain may facilitate the transitioning of the Argonaute ternary complex into a cleavage active conformation. The MID domain may bind a 5'phosphate and a first nucleotide of the gDNA. In some embodiments, the MID domain comprises a 5’ phosphate binding site having at least 2 or all 3 of the 3 conserved residues in the KQK motif as shown in FIG. 24A. In some embodiments, a lysine (K) residue in the KQK motif is replaced by an arginine (R) . In some embodiments, a glutamine (Q) residue in the KQK motif is replaced by an asparagine (N) , or a positively charged residue, such as lysine (K) or . The target nucleic acid can remain bound to the Argonaute through many rounds of cleavage by means of anchorage of the 5'phosphate in the MID domain.
[0124]
The Argonaute protein can comprise a nucleic acid-binding domain. The nucleic acid-binding domain can comprise a region that contacts a nucleic acid. The nucleic acid-binding domain can bind DNA or RNA, or both DNA and RNA. In some embodiments, the Argonaute protein binds a DNA and cleaves the DNA. In some embodiments, the Argonaute protein binds a single-stranded gDNA and cleaves a double-stranded DNA. In some embodiments, the nucleic acid-binding domain comprises a PAZ domain, which can use its oligonucleotide-binding fold to secure the 3'end of the designed nucleic acid-targeting nucleic acid.
[0125]
The Argonaute can comprise a nucleic acid-cleaving domain, such as a PIWI domain. In some embodiments, the Ago comprises a PIWI domain comprising a nuclease active site. In some embodiments, the nuclease active site binds a divalent cation. In some embodiments, the nuclease active site binds two divalent cations. For example, a first divalent cation may initiate a nucleophilic attack and activate a water molecule, and a second divalent cation may stabilize the transition state and leaving group. In some embodiments, the Argonaute comprises a nuclease active site having at least 2 or all 3 of the 3 conserved residues in the DDE motif as shown in FIG. 24B. In some embodiments, an aspartic acid (D) residue in the DDE motif is replaced by a glutamic acid (E) . In some embodiments, a glutamic acid (E) residue in the DDE motif is replaced by an aspartic acid (D) . In some embodiments, the nuclease active site further comprises one or more basic residues, such as histidine, arginine, lysine or combinations thereof. The histidine, arginine and/or lysine may play a role in catalysis and/or cleavage. In some embodiments, the nuclease active site comprises four negatively charged, evolutionary conserved amino acids, such as aspartate-glutamate-aspartate-aspartate/histidine (DEDX, SEQ ID NO: 157) , which form a catalytic tetrad that binds two divalent metal ions (such as Mg 2+ ions) and cleave a target nucleic acid into products bearing a 3'hydroxyl and 5'phosphate group. In some embodiments, depending on the type of Ago, the method is carried out in the presence of a divalent metal ion, such as Mg 2+, at a concentration of at least about any one of 0.1 mM , 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, 20 mM or more. Cleavage of the target nucleic acid by Argonaute can occur at a single phosphodiester bond in one strand, or at two phosphodiester bonds in both strands of the target nucleic acid. In some embodiments, the Ago protein comprises a functional nuclease active site that does not contain the DDE or DEDX (SEQ ID NO: 157) motif. In some embodiments, the Ago protein does not comprise a functional nuclease active site.
[0126]
Exemplary Argonautes and their protein sequences are shown in Table 1 below. In some embodiments, the Argonaute is AaAgo, AfAgo, BaAgo, BgAgo, CbAgo, CcAgo, CpAgo, CsAgo, CuAgo, ExAgo, FpAgo, HaAgo, HbAgo, HkAgo, HlAgo, HmAgo, HpAgo, IbAgo, LyAgo, MaAgo, MfAgo, Migo, MkAgo, MlAgo, Ago, NgAgo, NpAgo, NtAgo, PhAgo, PlAgo, RbAgo, ScAgo, SeAgo, SsAgo, SyAgo, TbAgo, TcAgo, TeAgo, ToAgo, or a functional derivative thereof. In some embodiments, the Argonaute is NgAgo, or a functional derivative thereof. In some embodiments, the Argonaute is PhAgo, or a functional derivative thereof. In some embodiments, the Argonaute is MiAgo, or a functional derivative thereof. In some embodiments, the Argonaute is MaAgo, or a functional derivative thereof. In some embodiments, the Argonaute is not TtAgo or RsAgo. In some embodiments, the Argonaute is not SeAgo. Argonaute proteins suitable for use in the methods described herein have been described, for example, in WO2017/107898, incorporated herein by reference in its entirety.
[0127]
Table 1. Exemplary Argonaute protein sequences.
[0128]
[0129]
[0130]
[0131]
In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to wild-type Argonautes of Table 1 (e.g., NgAgo) in the MID domain, PAZ domain, and/or PIWI domain. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to wild-type Argonautes of Table 1 (e.g., NgAgo, MiAgo, MaAgo, or PhAgo) in the MID domain, PAZ domain, and/or PIWI domain. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to a sequence selected from SEQ ID NOs: 1-42. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to a sequence selected from SEQ ID NOs: 1-42. In some embodiments, the Ago protein has a sequence selected from SEQ ID NOs: 1-42. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to SEQ ID NO: 1. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to SEQ ID NO: 1. In some embodiments, the Ago protein has SEQ ID NO: 1. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to SEQ ID NO: 2. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to SEQ ID NO: 2. In some embodiments, the Ago protein has SEQ ID NO: 2. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to SEQ ID NO: 11. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to SEQ ID NO: 11. In some embodiments, the Ago protein has SEQ ID NO: 11. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or 100%sequence homology to SEQ ID NO: 41. In some embodiments, the Argonaute protein comprises a sequence having at least about any one of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more, or about 100%sequence identity to SEQ ID NO: 41. In some embodiments, the Ago protein has SEQ ID NO: 41.
[0132]
In some embodiments, the Argonaute is a modified form of a wildtype Ago protein in Table 1. The modified form of the wild type Argonaute can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nuclease activity of the Argonaute. For example, the modified Argonaute can have less than about any one of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%or less of the nuclease activity of the wild-type Argonaute (e.g., NgAgo, MiAgo, MaAgo, or PhAgo) . The modified form of the Argonaute can have no substantial nuclease activity. For example, one or more of the conserved residues in the DDE motif in the nuclease active site of the wildtype Ago can be mutated, e.g., to alanine, to provide an Argonaute that has no nuclease activity. One skilled in the art will recognize that mutations other than alanine substitutions are suitable. In some embodiments, sequences can be inserted to an Argonaute protein to reduce its activity. In some embodiments, the modified form of the wild type Argonaute can have more than about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more nuclease activity of the wild-type Argonaute (e.g., NgAgo, MiAgo, MaAgo, or PhAgo) .
[0133]
Unless specified otherwise, “Argonaute” or “Ago” may refer to the polypeptide (s) corresponding to a wildtype Argonaute protein or a functional derivative thereof, or the polynucleotide (s) encoding the polypeptide. “Functional derivative” refers to a modified form of a protein having substantially the same function or activity as the wildtype protein, but comprising one or more changes to the amino acid sequence or chemical composition, including, but not limited to, mutations (e.g., deletion, insertion, substitution, etc. ) , non-natural amino acid variants, fusions to other polypeptides (e.g., affinity tags, signal peptides, etc. ) , conjugation to non-amino acid moieties (e.g., dyes) , and a chimeric protein having other functional modalities.
[0134]
In some embodiments, the Ago protein is a fusion protein. A fusion protein can comprise one or more of the same non-native sequences not naturally found in the wildtype Ago. In some embodiments, the Ago protein is fused to one or more affinity tags, such as HA and FLAG tags, which can facilitate purification of the Ago protein. In some embodiments, the Ago protein is fused to a fluorescent protein, such as GFP or RFP, for visualization or tracking of the Ago protein inside cells. In some embodiments, the Ago protein is fused to a self-penetrating peptide to facilitate intracellular delivery of the Ago-gDNA complex. In some embodiments, the Ago protein is fused to a subcellular localization signal of the Ago, e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like. The fusion moiety (such as affinity tag, fluorescent protein, self-penetrating peptide, and/or localization signal) may be fused to either the N-terminus, or the C-terminus, or both termini of the Ago protein using standard recombinant methods known in the art.
[0135]
The Argonaute proteins described herein can be expressed recombinantly in the host cell, i.e., the cell containing the target nucleic acid to be modified by the Ago protein. The nucleic acid encoding the Argonaute protein can be isolated and sequenced from any of the species listed in Table 1. Alternatively, an Argonaute coding sequence can be designed based on a naturally occurring Ago sequence, such as any one of SEQ ID No: 1-42, and a nucleic acid having the designed sequence can be synthesized using nucleotide synthesizer or PCR techniques. In some embodiments, the Ago coding sequence is further engineered, such as by site-directed mutagenesis, for expression of a functional derivative or mutant variant of the wildtype Argonaute protein. In some embodiments, the nucleic acid comprising an Ago coding sequence fused to one or more additional component, such as a signal sequence. In some embodiments, for nuclear expression of the Ago protein in the host cell, a nuclear localization sequence is fused to the N-terminus of the Ago sequence.
[0136]
In some embodiments, the nucleic acid encoding the Ago protein is codon optimized for expression in host cells, such as eukaryotic cells. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA) , which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" , and these tables can be adapted in a number of ways. See Nakamura, Y., et al. ″Codon usage tabulated from the international DNA sequence databases: status for the year 2000, " Nucl. Acids Res. 28: 292 (2000) . Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA) , are also available.
[0137]
In some embodiments, the nucleic acid encoding the Ago protein is subcloned into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a host cell. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transfected with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. In some embodiments, a vector for expression of the Ago protein in prokaryotic cells is provided. In some embodiments, a vector for expression of the Ago protein in eukaryotic cells, such as mammalian cells, is provided.
[0138]
A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
[0139]
In some embodiments, the vector is a plasmid. Examples of plasmids include, but are not limited to, pGEX6P-1, and pcDNA3.1. In some embodiments, the plasmid is supercoiled. In some embodiments, the plasmid is ultrapure. As used herein, “ultrapure” plasmid refers to plasmid preparations that is substantially free from contamination by endotoxins or non-viral microorganisms, and the plasmid is at least about any one of 90%, 95%, 97%, 99%, or more supercoiled. Ultrapure plasmids can be prepared using commercial plasmid purification kits. Supercoiling of a plasmid can be determined by gel electrophoresis.
[0140]
In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vector, retroviral vectors, vaccinia vector, herpes simplex viral vector, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals.
[0141]
A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the host cell in vitro. In some embodiments, adenovirus vectors are used. In some embodiments, lentivirus vectors are used. In some embodiments, self-inactivating lentiviral vectors are used.
[0142]
In some embodiments, a vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. Additional components of the vector may include, but are not limited to, a ribosome binding site (RBS) , a signal sequence, and a transcription termination sequence. In general, vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
[0143]
In some embodiments, the nucleic acid is operably linked to a promoter. A large number of promoters recognized by a variety of potential host cells are well known. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter. Inducible promoter is a promoter that initiates increased levels of transcription of the coding sequence under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.
[0144]
Promoters suitable for use with prokaryotic host cells include the phoA promoter, -lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the Ago protein.
[0145]
Polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2) , bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40) , from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
[0146]
In some embodiments, the vector for expression in higher eukaryotes comprises an enhancer sequence. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) , or from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) , the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′or 3′to the polypeptide encoding sequence, but is preferably located at a site 5′from the promoter.
[0147]
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. For example, at the 3′end of most eukaryotic is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′end of the coding sequence. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the polypeptide-encoding mRNA. All of these sequences may be inserted into eukaryotic expression vectors.
[0148]
In some embodiments, the nucleic acid encoding the Ago protein is a RNA. In some embodiments, the nucleic acid is a RNA vector. In some embodiments, the nucleic acid is an mRNA encoding the Ago protein. In some embodiments, the mRNA encoding the Ago protein comprises one or more modifications to enhance its stability, enhance its expression, and/or reduce its immunogenicity. In some embodiments, the mRNA comprises a modified backbone and/or modified internucleoside linkages. In some embodiments, the mRNA comprises one or more phosphorothioate linkages. In some embodiments, the mRNA comprises one or more modified nucleobases, such as 5-methyl cytosine and/or pseudouridine. In some embodiments, the mRNA comprises a 5’ cap.
[0149]
Further provided herein are nucleic acids and vectors (such as plasmids or viral vectors) encoding any one of the Argonaute proteins described herein, and use of any one of the nucleic acids and vectors for expression of the Ago proteins, or for gene editing, including, but not limited to, modification of target nucleic acids, targeted editing of intracellular viral DNA, and targeted editing of chromosomal DNA. Also provided is use of any one of the nucleic acids and vectors in preparation of a gene editing kit or an analytic or interference agent.
[0150]
B. Guide DNA
[0151]
The Argonautes described herein uses a guide DNA that is a single-stranded oligonucleotide DNA having a sequence designed to be perfectly complementary or substantially complementary to the target nucleic acid to be modified. In some embodiments, a single guide DNA is required for the Ago protein to induce a double-strand break in a double-stranded target nucleic acid. In some embodiments, two guide DNAs each targeting an opposite strand of a double-stranded target nucleic acid are required for the Ago protein to induce a double-strand break in the target nucleic acid. In some embodiments, the Ago protein uses a plurality of guide DNAs targeting different sequences in the target nucleic acid.
[0152]
In some embodiments, the Ago protein and the guide DNA (s) do not naturally occur together. In some embodiments, the guide DNA comprises a sequence that does not substantially hybridize to any part of the genomic sequence of the host cell. In some embodiments, the guide DNA comprises a modification, such as 5’ phosphorylation, or modification to the base or backbone portion of a nucleotide, that does not naturally occur in the host cell.
[0153]
In some embodiments, the Ago protein does not have any preference for specific sequence or nucleotides in the guide DNA. In some embodiments, the 5’ terminus of the guide DNA is phosphorylated. 5’ phosphorylated guide DNA can be prepared by chemical synthesis, or by phosphorylating an oligonucleotide DNA using a kinase, such as T4 PNK. In some embodiments, the 5’ phosphorylated guide DNA is produced endogenously by a bacterial cell. For example, a plasmid can be transfected into the bacterial cell to produce 5’ phosphorylated guide DNAs targeting sequences derived from the plasmid. In some embodiments, the plasmid is a linearized plasmid. In some embodiments, the gDNA is substantially purified, such as at least about any one of 80%, 85%, 90%, 95%, 99%, or more pure. Guide DNA can be purified by known methods in the art, such as HPLC, gel electrophoresis, or using DNA purification kits.
[0154]
The length of the guide DNA may influence the activity (such as specific binding and/or nuclease activity) of the Ago-gDNA complex. In some embodiments, the guide DNA has about 10 to about 50 nucleotides ( “nt” ) , such as any one of about 10 nt to about 20 nt, about 20 nt to about 30 nut, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 15 nt to about 30 nt, about 20 nt to about 40 nt, about 15 nt to about 25 nt, or about 20 nt to about 35 nt. In some embodiments, the guide DNA has about any one of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the guide DNA has about 20 nucleotides to about 27 nucleotides, or about 23 nucleotides to 25 nucleotides. In some embodiments, wherein the Ago protein is derived from NgAgo, the guide DNA is 24 nucleotides long. The optimal length of the gDNA may vary depending on the species from which the Ago protein is derived, and can be determined by a skilled person in the art using gDNAs of different length in an in vitro or in cell activity assay, such as binding, plasmid cleavage, or reporter gene (e.g., GFP) silencing.
[0155]
In some embodiments, the activity (such as specific binding and/or nuclease activity) of the Ago-gDNA complex is sensitive to nucleotide mismatches between the gDNA and the target locus. In some embodiments, the efficiency of modification or cleavage of the target locus is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more when the gDNA comprises one mismatch to the target locus. In some embodiments, the efficiency of modification or cleavage of the target locus is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more when the gDNA comprises two mismatches, such as two consecutive mismatches, to the target locus. In some embodiments, the efficiency of modification or cleavage of the target locus is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more when the gDNA comprises three mismatches, such as three consecutive mismatches, to the target locus. In some embodiments, the gDNA is perfectly complementary to the target locus. In some embodiments, the gDNA has one mismatch to the target locus. In some embodiments, the gDNA has two mismatches to the target locus. In some embodiments, the gDNA has three mismatches to the target locus. In some embodiments, the gDNA does not have a mismatch in any one of positions 8, 9, 10, or 11 from the 5’ terminus, compared to the sequence of the target locus. In some embodiments, the gDNA does not have any three consecutive nucleotides that are mismatches to the target locus.
[0156]
In some embodiments, the guide DNA comprises a modified backbone and/or modified internucleoside linkages. In some embodiments, the guide DNA comprises one or more phosphorothioate linkages. Various salts (e.g., potassium chloride or sodium chloride) , mixed salts, and free acid forms can also be included. In some embodiments, the guide DNA comprises one or more modified nucleobases.
[0157]
C. Ago-gDNA complex
[0158]
In some embodiments, the Ago protein and the gDNA form a complex in the cell. Generally, the Ago protein and the gDNA bind to each other in a molar ratio of about 1: 1. As used herein, binding of the Ago protein to gDNA to form an Ago-gDNA complex is referred herein as “loading” of gDNA to the Ago protein. In some embodiments, the Ago-gDNA complex follows a “one-guide faithful” rule, i.e., the Ago protein does not dissociate from the bound gDNA in the complex, or switch a gDNA in a loaded Ago-gDNA complex with a free, unbound gDNA. In some embodiments, the Ago protein does not dissociate from a bound gDNA at a temperature lower than about any one 40℃, 45℃, 50℃, or 55℃. In some embodiments, the Ago protein does not dissociate from a bound gDNA or bind to a free gDNA at about 37℃ over an incubation period of at least about any one of 4, 8, 12, 16, 24, or more hours. In some embodiments, a pre-formed Ago-gDNA complex does exchange the gDNA with a different gDNA in the cell.
[0159]
In some embodiments, the Ago-gDNA complex is prepared by expressing the Ago protein in the presence of the gDNA. In some embodiments, a nucleic acid (such as a vector) encoding the Ago protein and a gDNA are co-transfected into a host cell to provide the Ago-gDNA complex. In some embodiments, the host cell is a cell that does not have endogenous 5’ phosphorylated gDNA. In some embodiments, the host cell is a mammalian cell, such as 293T cell or HeLa cell. In some embodiments, the host cell is a bacterial cell, such as E. coli. In some embodiments, a nucleic acid (such as a plasmid) encoding the Ago protein and a linearized vector encoding the gDNA are co-transfected into a bacterial cell to provide the Ago-gDNA complex.
[0160]
D. Target nucleic acid
[0161]
The methods disclosed herein are applicable for a variety of target nucleic acids. In some embodiments, the target nucleic acid is a DNA. In some embodiments, the target nucleic acid is a RNA, such as mRNA. In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the target nucleic acid comprises both single-stranded and double-stranded regions. In some embodiments, the target nucleic acid is linear. In some embodiments, the target nucleic acid is circular. In some embodiments, the target nucleic acid comprises one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotides analogs. In some embodiments, the target nucleic acid is not modified.
[0162]
The target nucleic acid may be of any length, such as about at least any one of 100 bp, 200 bp, 500 bp, 1000 bp, 2000 bp, 5000 bp, 10 kb, 20 kb, 50 kb, 100 kb, 200 kb, 500 kb, 1Mb, or longer. The target nucleic acid may also comprise any sequence. In some embodiments, the target nucleic acid is GC-rich, such as having at least about any one of 40%, 45%, 50%, 55%, 60%, 65%, or higher GC content. In some embodiments, the target nucleic acid is not GC-rich. In some embodiments, the target nucleic acid has one or more secondary structures or higher-order structures. In some embodiments, the target nucleic acid is not in a condensed state, such as in a chromatin, to render the target locus inaccessible by the Ago-gDNA complex.
[0163]
In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the target nucleic acid is present in the nucleus of the cell. In some embodiments, the target nucleic acid is endogenous to the cell. In some embodiments, the target nucleic acid is a genomic DNA. In some embodiments, the target nucleic acid is a chromosomal DNA. In some embodiments, the target nucleic acid is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5’ or 3’ untranslated region, etc. In some embodiments, the target nucleic acid is a non-coding gene, such as transposon, miRNA, tRNA, ribosomal RNA, ribozyme, or lincRNA. In some embodiments, the target nucleic acid is a plasmid.
[0164]
In some embodiments, the target nucleic acid is exogenous to a cell. In some embodiments, the target nucleic acid is a viral nucleic acid, such as viral DNA or viral RNA. In some embodiments, the target nucleic acid is a horizontally transferred plasmid. In some embodiments, the target nucleic acid is integrated in the genome of the cell. In some embodiments, the target nucleic acid is not integrated in the genome of the cell. In some embodiments, the target nucleic acid is a plasmid in the cell. In some embodiments, the target nucleic acid is present in an extrachromosomal array.
[0165]
The target locus is a segment of the target nucleic acid that hybridizes to the gDNA. In some embodiments, the target locus is cleaved by the Ago-gDNA complex. In some embodiments, the target nucleic acid has only one copy of the target locus. In some embodiments, the target nucleic acid has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target locus. For example, a target locus comprising a repeated sequence in a genome of a viral nucleic acid or a bacterium may be targeted by an Ago-gDNA to inhibit or kill the virus or the bacterium.
[0166]
In some embodiments, the target locus is a DNA locus. In some embodiments, the target locus is a RNA locus. In some embodiments, the target locus is double stranded. In some embodiments, the target locus is single-stranded. In some embodiments, the target locus is a double-stranded DNA.
[0167]
The target locus may comprise any sequence, as the Ago protein has no preferences to bind a particular sequence or sequence motif. In some embodiments, the target locus is GC rich. In some embodiments, the target locus has a GC content of at least about any one of 40%, 50%, 60%, 70%, 80%, or more. In some embodiments, the target locus is a GC-rich fragment in a non-GC-rich target nucleic acid. In some embodiments, the target locus is present in a readily accessible region of the target nucleic acid. In some embodiments, the target locus is in an exon of a target gene. In some embodiments, the target locus is across an exon-intron junction of a target gene. In some embodiments, the target locus is present in a non-coding region, such as a regulatory region of a gene. In some embodiments, wherein the target nucleic acid is exogenous to a cell, the target locus comprises a sequence that is not found in the genome of the cell.
[0168]
In some embodiments, the target nucleic acid, and/or the target locus includes a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target nucleic acids include disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation (s) or genetic variation (s) that is directly responsible or is in linkage disequilibrium with a gene (s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. Mutations in these genes and pathways can result in production of improper proteins or proteins in improper amounts which affect function. In some embodiments, the target locus is a disease-associated locus. In some embodiments, the target locus comprises a mutation or genetic variation in a disease-associated gene. Examples of disease-associated genes and polynucleotides, and disease-associated loci are available from MeKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md. ) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) , available on the World Wide Web.
[0169]
The present application contemplates methods of generating isogenic lines of cells of mammalian cells for the study of genetic variations in a disease. In some embodiments, the method provides a single-nucleotide substitution at the target locus, which can be used to study the effect of single nucleotide polymorphisms. The present application also contemplates genome modification of microbes, cells, plants, animals or synthetic organisms for the generation of biomedically, agriculturally, and industrially useful products. The methods may be used as a biological research tool, for understanding the genome, e.g. gene knockout or knock-in studies. The methods may also be used as a therapeutic for targeting specific strains of bacterial infections, or viral infection.
[0170]
E. Donor DNA
[0171]
In some embodiments, the method comprises contacting the target nucleic acid with a donor DNA comprising one or more homologous sequences to the target nucleic acid. Without being bound by any theory of hypothesis, double-strand breaks in a target nucleic acid induced by the Ago-gDNA complex can initiate or stimulate the endogenous Homology Directed Recombination (HDR) repair pathway in the cell, which integrates the donor DNA into the cleavage target locus. In some embodiments, the donor DNA comprises a 5’ homology arm, a 3’ homology arm, and an exogenous sequence to the cell that is disposed in between the 5’ homology arm and the 3’ homology arm. In some embodiments, the homology sequence, such as homology arm (s) , comprise a sequence that is at least about any one of 80%, 85%, 90%, 95%, 99%, or more, or 100%identical to the sequence flanking the cleavage site in the target nucleic acid. In some embodiments, the homology sequence, such as homology arm (s) , is at least about any one of 10, 20, 30, 40, 50, 100, or more nucleotides long. In some embodiments, the donor DNA comprises a substitution sequence of the target nucleic acid encompassing the target locus. In some embodiments, the substitution sequence differs from the sequence of the target nucleic acid by no more than about any of 10, 5, 4, 3, 2, or 1 nucleotide (s) .
[0172]
In some embodiments, the donor DNA comprises a sequence encoding a selection marker. The selection marker can be used to select cells having the donor DNA integrated in the target locus. Exemplary selection markers include, but are not limited to, proteins that: (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, tetracycline, hygromycin, and G418, (b) complement auxotrophic deficiencies, and (c) supply critical nutrients not available from complex media. In some embodiments, a drug, such as G418, is used in a selection regimen to arrest the growth, or kill cells that do not have the donor DNA integrated in the target locus. Those cells that are successfully modified with the donor DNA produce a protein conferring drug resistance and thus survive the selection regimen. Examples of selectable markers suitable for mammalian cells also include DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
[0173]
In some embodiments, the selection marker is a reporter protein that allows selection by confirming expression of the reporter protein. Examples of reporter proteins include, but are not limited to, glutathione-S-transferase (GST) , horseradish peroxidase (HRP) , chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP, e.g., eGFP) , HcRed, DsRed, cyan fluorescent protein (CFP) , yellow fluorescent protein (YFP) , and autofluorescent proteins including blue fluorescent protein (BFP) . In some embodiments, the donor DNA does not comprise a promoter for the reporter protein. Thus, the reporter protein is only expressed when the donor DNA is integrated in frame with an endogenous gene of the cell.
[0174]
The donor DNA may be of any suitable length, such as at least about any one of 100bp, 200bp, 300bp, 500bp, 1kb, 2kb, 5kb, 10kb, or longer. The donor DNA may be prepared by chemical synthesis or PCR amplification from a template. In some embodiments, the donor DNA is substantially purified, such as at least about any one of 80%, 85%, 90%, 95%, 99%, or more pure. In some embodiments, the donor DNA is present in a vector. In some embodiments, the donor DNA is present in a plasmid, such as an ultrapure plasmid, or a linearized plasmid. The donor DNA can be purified by known methods in the art, such as HPLC, gel electrophoresis, or using DNA purification kits.
[0175]
F. Cell
[0176]
The methods described herein can be used to modify target nucleic acids in a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments the cell is in cell culture. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism, and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.
[0177]
In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the bacterial cell is not related to the bacterial species from which the Ago protein is derived. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell. In some embodiments, the cell is an eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.
[0178]
In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va. ) ) . In some embodiments, a cell transfected with one or more nucleic acids (such as Ago-coding vector and gDNA) or Ago-gDNA complex described herein is used to establish a new cell line comprising one or more vector-derived sequences to establish a new cell line comprising modification to the target nucleic acid. In some embodiments, cells transiently or non-transiently transfected with one or more nucleic acids (such as Ago-coding vector and gDNA) or Ago-gDNA complex described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
[0179]
In some embodiments, the cell is a primary cell. For example, cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. In some embodiments, the primary cells are harvest from an individual by any known method. For example, leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution can generally be a balanced salt solution, (e.g. normal saline, phosphate-buffered saline (PBS) , Hank's balanced salt solution, etc. ) , conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration. Buffers can include HEPES, phosphate buffers, lactate buffers, etc. Cells may be used immediately, or they may be stored (e.g., by freezing) . Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10%DMSO, 50%serum, 40%buffered medium) , and/or some other such common solution used to preserve cells at freezing temperatures.
[0180]
In some embodiments, the cell is an immune cell, such as T cells, Natural killer cells, and macrophages. In some embodiments, the cell is a human T cell obtained from a patient or a donor. The methods provided herein can be used to modify a target nucleic acid in a primary T cell for use in immunotherapy.
[0181]
In some embodiments, the cell is a stem cell or progenitor cell. Cells can include stem cells (e.g., adult stem cells, embryonic stem cells, iPS cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc. ) . Cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc.
[0182]
In some embodiments, the cell is a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
[0183]
In some embodiments, the cell is free or substantially free from contamination. Contaminations include endotoxins, chelating agents (such as EDTA) , and micro-organisms, such as mycoplasma, chlamydia, archaea, protozoa, and fungi. In some embodiments, the Ago-gDNA is sensitive to contamination of intracellular bacteria such as mycoplasma. Intracellular bacteria can be widespread and leave no visible signs of presence in cell lines. Intracellular bacteria should be carefully excluded from the cells before carrying out the methods described herein. In some embodiments, the cell, such as a cell line obtained from a commercial source, is treated with one or more antibiotics, such as penicillin and streptomycin, to remove contamination by micro-organisms. In some embodiments, the cell culture medium is heat inactivated to remove contamination by micro-organisms. In some embodiments, chelators, such as EDTA, is avoided in buffers for detaching and seeding cells into plates. In some embodiments, depending on the cell type, a divalent ion, such as Mg 2+, is supplemented to the cell at a concentration of at least about any one of 0.1 mM , 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or more.
[0184]
In some embodiments, the Argonaute induces double-stranded breaks or single-stranded breaks in a nucleic acid, (e.g. genomic DNA) . The double-stranded break can stimulate cellular endogenous DNA-repair pathways, including Homology Directed Recombination (HDR) , Non-Homologous End Joining (NHEJ) , or Alternative Non-Homologues End-Joining (A-NHEJ) . NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can result in deletion or insertion of one or more nucleotides at the target locus. HDR can occur with a homologous template, such as the donor DNA. The homologous template can comprise sequences that are homologous to sequences flanking the target nucleic acid cleavage site. In some cases, HDR can insert an exogenous polynucleotide sequence into the cleave target locus. The modifications of the target DNA due to NHEJ and/or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene knock-in, gene disruption, and/or gene knock-outs.
[0185]
In some embodiments, the cell culture is synchronized to enhance the efficiency of the methods. In some embodiments, cells in S and G2 phases are used for HDR-mediated gene editing. In some embodiments, the cell can be subjected to the method at any cell cycle. In some embodiments, cell over-plating significantly reduces the efficacy of the method. In some embodiments, the cell is at log growth phase at the time of the transfection of the ssDNA and/or the vector encoding the DNA-guided nuclease (such as Ago) . In some embodiments, the cell has a confluency of about 30%-80% (such as about any one of 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 30%-50%, 40%-60%, 60%-80%, or 40%-60%) at the time of the transfection. In some embodiments, the method is applied to a cell culture at no more than about any one of 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80%confluency.
[0186]
In some embodiments, binding of the Ago-gDNA complex to the target locus in the cell recruits one or more endogenous cellular molecules or pathways other than DNA repair pathways to modify the target nucleic acid. For example, in some embodiments, the Ago-gDNA complex recruits the RISC complex to silence an mRNA. In some embodiments, catalytically inactive Ago can be used to silence a target mRNA. In some embodiments, binding of the Ago-gDNA complex blocks access of one or more endogenous cellular molecules or pathways to the target nucleic acid, thereby modifying the target nucleic acid. For example, binding of the Ago-gDNA complex may block endogenous transcription or translation machinery to decrease the expression of the target nucleic acid.
[0187]
G. Cell assessment and selection
[0188]
In some embodiments, the cell is cultured for at least about any one of 8 hours, 12 hours, 24 hours, 48 hours, 60 hours, 3 days, 4 days, 6 days, or more, such as about 48 hours to 60 hours, after transfection of the nucleic acid encoding the Ago protein and the gDNA, and optionally the donor DNA into the cell. In some embodiments, the cell is cultured in a medium containing at least about 2%, such as about any one of 5%, 6%, 7%, 8%, 9%, or 10% (v/v) serum, at least about 6 hours after transfection of the gDNA. In some embodiments, the cell is allowed to grow to no more than about any one of 95%, 90%, 85%, or 80%confluency before assessment and/or selection.
[0189]
In some embodiments, the cell is selected based on one or more features, including, but not limited to expression of selection markers (e.g., antibiotic resistance protein, fluorescent tag, etc. ) , expression level of target nucleic acid, phenotypic change to the cell, or sequence of the target nucleic acid (e.g., a PCR amplicon of the target locus) . In some embodiments, a single clone having successful modification of the target nucleic acid is isolated.
[0190]
In some embodiments, a phenotypic change to the cell is assessed. In some embodiments, wherein an exogenous gene is knocked-in to the cell, a phenotypic change associated with the exogenous gene is assessed to select cells having successful integration of the exogenous gene. In some embodiments, wherein a mutation is introduced to a gene, a phenotypic change associated with the mutation or the gene is assessed. For example, if the gene is involved in development, a developmental defect in the cell or the organism derived from the cell that is associated with the mutation may be assessed to identify cells having successful introduction of the mutation. Other exemplary phenotypic changes suitable for screening include growth rate of the cell, cell cycle progression, and metabolic phenotypes. Phenotypic changes can be determined using known assays chosen for the target nucleic acid and its associated phenotype, including, for example, microscopy.
[0191]
In some embodiments, the expression level of the target nucleic acid is assessed. Expression levels can be determined at either the RNA level or the protein level for a gene. Many methods are known in the art to assess expression levels, including, but not limited to, Western blots and immunostaining for protein levels, and quantitative RT-PCR, RNAseq, and in situ hybridization for RNA levels. In some embodiments, the expression level is decreased by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
[0192]
In some embodiments, wherein the donor DNA comprises a selection marker, the expression level of the selection marker is assessed. In some embodiments, wherein the selection marker is an antibiotic resistance gene, the appropriate antibiotic is supplemented to the cell, e.g., at about 48 to 60 hours after the delivery of the Ago-gDNA complex or the Ago-encoding gene with the gDNA into the cell, to select for cells having successful integration of the donor DNA into the target nucleic acid. In some embodiments, wherein the selection marker is a fluorescent protein (e.g., mRFP, eGFP, etc. ) , expression of the selection marker is assessed by fluorescence microscopy, or by Fluorescence-assisted cell sorting (FACS) .
[0193]
In some embodiments, the target locus is amplified and assessed to select a cell having the desired mutation, or knock-in of exogenous sequence. PCR Primers can be designed to amplify regions of modification in the target nucleic acid, for example, at junctions between the target locus and the inserted sequence from the donor DNA, to provide amplicons for analysis. In some embodiments, the PCR amplicons can be analyzed by gel electrophoresis, restriction digestion, or by sequencing (such as Sanger sequencing) , to confirm that the target locus is modified as designed.
[0194]
IV. Kits and articles of manufacture
[0195]
The present application also provides compositions, kits and articles of manufacture for carrying out any one of the methods described herein.
[0196]
In some embodiments, there is provided a kit comprising: (a) a first composition comprising a single-stranded guide DNA and a first transfection agent (e.g., 2000) ; and (b) a second composition comprising a nucleic acid encoding a DNA-guided nuclease and a second transfection agent (e.g., 3000) , wherein the Ago protein and the guide DNA form a complex that is capable of specifically recognizing a target locus, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA.
[0197]
In some embodiments, there is provided a kit comprising: (a) a first composition comprising a single-stranded guide DNA and a first transfection agent (e.g., 2000) ; and (b) a second composition comprising a nucleic acid encoding an Ago protein and a second transfection agent (e.g., 3000) , wherein the Ago protein and the guide DNA form a complex that is capable of specifically recognizing a target locus at a temperature of about 10℃ to about 60℃, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA.
[0198]
In some embodiments, there is provided a kit comprising: (a) a first composition comprising a single-stranded guide DNA (such as a 5’ phosphorylated single-stranded guide DNA) and a first transfection agent (e.g., 2000) ; and (b) a second composition comprising a nucleic acid encoding an Ago protein derived from Natronobacterium gregoryi and a second transfection agent (e.g., 3000) , wherein the Ago protein and the guide DNA form a complex that is capable of specifically recognizing a target locus at a temperature of about 10℃ to about 60℃, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, the Ago protein comprises an amino acid sequence having at least about 80% (such as at least about any of 85%, 90%, 95%, 98%, 99%or more sequence homology, or about 100%sequence identity) sequence homology to SEQ ID NO: 1.
[0199]
In some embodiments, the nucleic acid encoding the Ago protein is operably linked to a promoter. In some embodiments, the nucleic acid encoding the Ago protein is present in a vector, such as a viral vector. In some embodiments, the vector is an ultrapure plasmid. In some embodiments, the nucleic acid encoding the Ago protein is an mRNA. In some embodiments, the nucleic acid encoding the Ago protein is codon-optimized for a species of interest. In some embodiments, the Ago protein and the guide DNA forms a complex that is capable of specifically recognizing a target locus at a temperature of about 10℃ to about 60℃ (such as about 37℃) , and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA. In some embodiments, the Ago protein is capable of cleaving the target locus. In some embodiments, wherein the target locus is a double-stranded DNA, the Ago protein is capable of inducing a double-strand break in the target locus. In some embodiments, the Ago protein is not capable of cleaving the target locus. In some embodiments, the guide DNA does not dissociate from the Ago protein at a temperature lower than about 50℃. In some embodiments, the guide DNA is about 10 to about 50 nucleotides (such as about 20 to about 30 nucleotides) long. In some embodiments, the sequence of the target locus comprises no more than about 3 mismatches (such as no mismatch) to the sequence of the guide DNA. In some embodiments, the target locus has a GC content of at least about 60%. In some embodiments, the molar ratio between the guide DNA and the Ago protein is at least about 1: 1. In some embodiments, the Ago protein comprises a nuclear localization signal (NLS) .
[0200]
In some embodiments, the first transfection agent is the same as the second transfection agent. In some embodiments, the first transfection agent is different from the second transfection agent. In some embodiments, the first transfection agent is 2000. In some embodiments, the first transfection agent is a DEAE (diethylaminoethyl) dextran. In some embodiments, the first transfection agent is HD. In some embodiments, the second transfection agent is 3000. In some embodiments, the first composition and/or the second composition has a pH of about 7.2-7.6 (such as about any one of 7.2, 7.3, 7.4, 7.5 or 7.6) . In some embodiments, the first composition and/or the second composition has no more than about 2% (v/v) of serum. In some embodiments, the medium is essentially free of serum.
[0201]
In some embodiments, the kit comprises a donor DNA. In some embodiments, the kit comprises a third composition comprising a donor DNA and a third transfection agent, such as 2000, DEAE (diethylaminoethyl) dextran, or HD.
[0202]
In some embodiments, the kit comprises one or more reagents for use in any one of the methods described herein. Reagents may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form) . A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is non-alkaline. In some embodiments, the buffer has a pH from about 7.2-7.6.
[0203]
In some embodiments, the kit further comprises instructions for carrying out any one of the methods described herein. The kits described herein can be used for modification of a target nucleic acid, genome editing, introducing mutations (e.g., indels, frameshift, knock-out or knock-in, and substitution) at a target locus, altering the phenotype of a cell, as analytic or interference agents. Also provided are articles of manufactures comprising any one of the kits described herein.
[0204]
EXAMPLES
[0205]
The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples are offered by way of illustration and not by way of limitation.
[0206]
Example 1: Transfection of single-stranded DNA (ssDNA)
[0207]
In this example, single-stranded DNA was transfected into mammalian cells under various conditions to identify an optimal condition for transfection of ssDNA into cells.
[0208]
1. Effect of medium pH on intracellular ssDNA distribution
[0209]
Alexa-488 labeled ssDNA packed with 2000 was transfected into 293T cells in Opti-MEM medium at a pH of 7.4 (newly opened medium bottle) or at a pH of 7.6 (medium previously opened and exposed to air) . After 6 hours, cells were examined by fluorescent microscopy and phase contrast microscopy. As shown in FIG. 1A, the ssDNA distributed evenly in the cytoplasm of cells cultured in DMEM at pH 7.4. In contrast, as shown in FIG. 1B, the ssDNA aggregated in a spotted pattern when cells were cultured in DMEM at pH 7.6. As shown in FIG. 1C, the arrows indicated accumulated ssDNA aggregates when cell were cultured in alkaline medium (i.e., pH 7.6) . Without being bound by any theory or hypothesis, the aggregates may sequester single-stranded gDNA, resulting in unavailability of the gDNA to load into an Ago-gDNA complex.
[0210]
Alkalized medium is a common, but neglected issue in the scientific community. When new bottles of Dulbecco's Modified Eagle Medium (DMEM) , Iscove's Modified Dulbecco's Medium (IMDM) , Eagle's Minimum Essential Medium (EMEM) or RPMI 1640 were opened for 10 minute on 2 consecutive days (for a total of 20 minutes) , the pH value of the media increased from 7.35-7.42 to 7.63-7.91. When the alkalized medium was placed in 5%CO 2 incubator for 24 hours in flasks with vent cap, the pH value reduced to 7.53-7.78, but such level was still above the physiological range of 7.35-7.45. A bottle of DMEM opened in less than a month but with frequent use had a pH of 8.2. According to the manufacturers, the culture media usually contain NaHCO 3, which is decomposed to CO 2 and evaporation of CO 2 increases PH, especially when the medium is warmed. As alkaline medium results in aggregation of transfected ssDNA in cells, transfection efficiency of single-stranded guide DNA is best achieved when contacting a cell cultured in a medium at pH of about 7.4-7.6. It is recommended that opened culture medium bottles should be sealed, for example, with parafilm. Also, exposure of the culture medium to air should be kept a minimum during experiments. Cell culture medium not used after 1 months of the opening date should be discarded.
[0211]
2. Effect of serum level in medium on ssDNA transfection efficiency
[0212]
Alexa-488 labeled 24nt-ssDNA was packed with 2000 in Opti-MEM (no serum) for 10 minutes. The mixture was then added to 293T or HeLa cells in the Opti-MEM medium containing 0-10% (v/v) fetal bovine serum (FBS) . The efficiency of ssDNA transfection was determined using flow cytometry 6 hours after transfection. As shown in FIGs. 2A amd 2B, the ssDNA transfection efficiency into both 293T cells and HeLa cells decreased as the serum level of the medium increased, suggesting that a higher transfection efficiency of the single-stranded guide DNA can be achieved using a cell culture medium having a low serum level at the time of the transfection.
[0213]
The effect of serum on ssDNA transfection efficiency was further investigated using different transfection agents. 100 ng Alexa-488 labeled 24-nt ssDNA was packed with 0.2 μl 2000, 0.15 μl 3000 (with 0.2 μl P3000) , 0.3 μl HD or 0.4 μl DEAE-Dextran according to the manufacturers’ instructions. The packed ssDNA was then added into each well of 293T cells in a 96-well plate in the Opti-MEM medium with or without 10%FBS. The efficiency of transfection was examined by flow cytometry 6 hours after transfection. As shown in FIG. 2C, the presence of serum in the cell culture medium at the time of ssDNA transfection significantly decreased the ssDNA transfection efficiency for all transfection reagents tested. Furthermore, 2000 yielded the highest ssDNA transfection efficiency.
[0214]
Next, the effect of serum supplement after ssDNA transfection on the stability of intracellular ssDNA was investigated. Alexa-488 labeled 24-nt ssDNA was transfected into 293T cells by 2000 in a culture medium without serum. After 6 hours, transfected cells in some wells were supplemented with serum (to a final concentration of 10%) . Degradation of ssDNA inside cells was monitored by flow cytometry. As shown in FIG. 2D, although serum in the medium impaired transfection efficiency, serum supplement to the culture medium after transfection reduced degradation of intracellular ssDNA. Without being bound by theory or hypothesis, the serum supplement may suppress autophagy in transfected cells. Thus, one strategy for gene-editing methods described herein is to transfect guide ssDNA into cells in a medium with 0-2%serum, and 6 hours after transfection, supplement the medium with serum to a final concentration of 10%.
[0215]
3. Effect of co-packing of ssDNA and plasmid on transfection efficiency
[0216]
Cells in each well of a 24-well plate were transfected with 330 ng of a plasmid coding a Flag-tagged NgAgo simultaneously with a 24nt single stranded guide DNA (ssDNA-1 and ssDNA-2 respectively) . The 24nt ssDNA was co-packed with the plasmid using 2000. The quantities of the ssDNA co-packed with the plasmid are indicated in FIG. 3A. The total amount of DNA transfected into the cells in each case did not exceed the manufacturer’s suggested amount, which is 500 ng using 2000. After 48 hours, cells were harvested and the expression levels of NgAgo were evaluated by Western blotting using an anti-Flag antibody. Results shown in FIGs. 3A demonstrate decreased NgAgo expression as the ssDNA quantity increased.
[0217]
In a second experiment, cells in each well of a 96-well plate were transfected with 50 ng GFP-expressing plasmid packed together or packed separately with 50 ng ssDNA by 2000.3 different 24-nt ssDNAs with or without 5’ phosphorylation were tested individually. After 24 hours, cells were harvested and the expression levels of GFP were determined using flow cytometry. The mean fluorescence intensity (MFI) was normalized against the MFI of cells transfected with GFP-expressing plasmid alone. As shown in FIG. 3B, co-packing of ssDNA with the plasmid significantly impaired the transfection efficiency of the plasmid, consequently reducing GFP expression levels.
[0218]
4. Dynamics of ssDNA distribution and plasmid expression after transfection
[0219]
Alexa-488 labeled ssDNA was transfected into cells, and the accumulation and decay of the ssDNA inside the cells were monitored by flow cytometry. As shown in FIG. 4A, the peak level of ssDNA inside cells appeared at around 6 hours after transfection. Afterwards, the level of intracellular ssDNA dropped quickly. At around 15 hours post-transfection, less than 20%of ssDNA was observed in the cells.
[0220]
In a second experiment, a GFP-expressing plasmid was transfected into cells, and the expression level of GFP was monitored by flow cytometry. As shown in FIG. 4B, the expression level of GFP reached the peak and plateaued between 24 hours and 36 hours after the transfection. This result suggests that intracellular gene-editing efficiency using NgAgo-gDNA may be optimized by first transfecting a plasmid encoding NgAgo followed by transfection of the gDNA (e.g., about 12 hours) , which will assure the availability of expressed NgAgo at the time of loading gDNA.
[0221]
Example 2: Genome editing in mammalian cells mediated by NgAgo-gDNA
[0222]
Non-Homologous End-Joining (NHEJ) is used to knock-in an eGFP-coding sequence (donor DNA) to a locus in exon 11 of the human DYRK1A gene. FIG. 5a shows a schematic of the genome-editing strategy. The G10 (SEQ ID NO: 51) guide DNA was used to target the locus.
[0223]
293T cells were cultured in a well of a 24-well plate. At log-phase of growth, 500 ng NgAgo-expressing plasmid was transfected into the cells using 3000.12 hours later, the cell culture medium was changed to a medium having 0-2%FBS. A double-stranded GFP-coding sequence and a single-stranded 24nt DNA (G10) were separately packed with 2000, and then pulsed into the cells. After another 36 hours, cells were harvested for analysis.
[0224]
Genomic DNA was extracted from the cells, purified and subjected to nested PCR reactions using primer pairs shown in FIG. 5B. For each PCR reaction, a primer for the primary genome (labeled as “DYRK1A” ) and a primer for the insert (labeled as “GFP” ) were used. The PCR products were then sequenced. Six different modified sequences were obtained and shown in FIG. 5B, in which the filled bars represent sequences matching the original genome or the GFP insert, and the non-filled bars indicate indels. Two representative sequencing chromatograms and sequences are shown in FIG. 5C. The sequencing analysis confirmed correct insertion of the donor eGFP DNA into the DYRK1A locus, and indel mutations at the eGFP-DYRK1A junction.

Claims

[Claim 1]
A method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent, wherein the medium has a pH of about 7.2-7.6.
[Claim 2]
The method of claim 1, wherein the medium has no more than about 2% (v/v) of serum.
[Claim 3]
A method of transfecting a single-stranded DNA into a cell, comprising contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent, wherein the medium has no more than about 2% (v/v) of serum.
[Claim 4]
The method of any one of claims 1-3, wherein the transfection agent is 2000.
[Claim 5]
The method of any one of claims 1-4, further comprising contacting the cell with a second composition comprising a second nucleic acid and a second transfection agent.
[Claim 6]
A method of modifying a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease and a first transfection agent and a second composition comprising a single-stranded guide DNA and a second transfection agent, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA, wherein the medium has a pH of about 7.2-7.6.
[Claim 7]
The method of claim 6, wherein the medium has no more than about 2% (v/v) of serum.
[Claim 8]
A method of modifying a target nucleic acid in a cell, comprising: contacting the cell in a medium with a first composition comprising a nucleic acid encoding a DNA-guided nuclease and a first transfection agent and a second composition comprising a single-stranded guide DNA and a second transfection agent, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA, wherein the medium has no more than about 2% (v/v) of serum.
[Claim 9]
A method of modifying a target nucleic acid in a cell, comprising: (a) transfecting a nucleic acid encoding a DNA-guided nuclease into the cell; and subsequently (b) transfecting a single-stranded guide DNA into the cell, wherein the DNA-guided nuclease and the guide DNA form a complex that specifically recognizes a target locus in the target nucleic acid, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA.
[Claim 10]
The method of claim 9, wherein the single-stranded guide DNA is transfected into the cell about 2 to about 48 hours after the transfection of the vector encoding the Ago protein.
[Claim 11]
The method of claim 9 or claim 10, wherein step (b) comprises contacting the cell in a medium with a composition comprising the single-stranded DNA and a transfection agent.
[Claim 12]
The method of claim 11, wherein the medium has a pH of about 7.2-7.6.
[Claim 13]
The method of claim 11 or 12, wherein the medium has no more than about 2% (v/v) of serum.
[Claim 14]
The method of any one of claims 2-5, 7-8 and 13, wherein the medium is essentially free of serum.
[Claim 15]
The method of any one of claims 2-5, 7-8 and 13-14, wherein the medium is supplemented with serum to a final concentration of about 5% (v/v) or more after at least about 2 hours of the contacting of the cell with the composition comprising the single-stranded DNA or the single-stranded guide DNA.
[Claim 16]
The method of any one of claims 1-15, wherein the cell has a confluency of about 30%-80%at the time of the contacting of the cell with the composition comprising the single-stranded DNA or the single-stranded guide DNA.
[Claim 17]
The method of any one of claims 10-16, wherein the transfection agent is 2000.
[Claim 18]
The method of any one of claims 6-17, wherein the DNA-guided nuclease is an Argonaute (Ago) protein.
[Claim 19]
The method of claim 18, wherein the Ago protein cleaves the target locus.
[Claim 20]
The method of claim 19, wherein the target locus is a double-stranded DNA, and wherein the Ago protein induces a double-strand break in the target locus.
[Claim 21]
The method of any one of claims 18-20, wherein the Ago protein is derived from an organism selected from the group consisting of Natronobacterium gregoryi, Microcystis aeruginosa, Halogeometricum pallidum, Natrialba asiatica, Natronorubrum tibetense, Natrinema pellirubrum, Halogeometricum borinquense, Thermococcus barophilus, Thermosynechococcus elongatus, Halorubrum lacusprofundi, Microcystis sp., Synechococcus sp., Clostridium bartlettii, Clostridium perfringens, Clostridium sartagoforme, Clostridium sp., Intestinibacter bartlettii, Ferroglobus placidus, Halobacterium sp., Methanocaldococcus fervens, Pseudomonas luteola, Thermogladius cellulolyticus, Aromatoleum aromaticum, Thermococcus onnurineus, Methanopyrus kandleri, Synechococcus elongatus, Anoxybacillus flavithermus, Exiguobacterium sp., Lyngbya sp., Clostridium butyricum, Halorubrum kocurii, Burkholderia ambifaria, Burkholderia graminis, Haloarcula marismortui, Mesorhizobium loti, Rhodobacterales bacterium and Pedobacter heparinus.
[Claim 22]
The method of claim 21, wherein the Ago protein is derived from Natronobacterium gregoryi.
[Claim 23]
The method of any one of claims 18-21, wherein the Ago protein comprises an amino acid sequence having at least about 80%sequence homology to a sequence selected from the group consisting of SEQ ID NOs: 1-42.
[Claim 24]
The method of claim 22, wherein the Ago protein comprises an amino acid sequence having at least about 80%sequence homology to SEQ ID NO: 1.
[Claim 25]
The method of any one of claims 6-24, wherein the sequence of the target locus comprises no more than about 3 mismatches to the sequence of the guide DNA.
[Claim 26]
The method of any one of claims 6-25, wherein the target locus has a GC content of at least about 60%.
[Claim 27]
The method of any one of claims 6-26, wherein the guide DNA is phosphorylated at the 5’ terminus.
[Claim 28]
The method of any one of claims 6-27, wherein the cell is transfected with the guide DNA for at least two times.
[Claim 29]
The method of any one of claims 6-28, wherein the nucleic acid encoding the DNA-guided nuclease is present in a vector.
[Claim 30]
The method of claim 29, wherein the vector is an ultrapure plasmid.
[Claim 31]
The method of any one of claims 6-30, wherein the target nucleic acid is endogenous to the cell.
[Claim 32]
The method of claim 31, wherein the target nucleic acid is a genomic DNA.
[Claim 33]
The method of any one of claims 6-30, wherein the target nucleic acid is exogenous to the cell.
[Claim 34]
The method of claim 33, wherein the target nucleic acid is a viral DNA.
[Claim 35]
The method of any one of claims 1-34, wherein the cell is a prokaryotic cell.
[Claim 36]
The method of any one of claims 1-34, wherein the cell is a eukaryotic cell.
[Claim 37]
The method of any one of claims 18-36, wherein the modifying comprises site-specific cleavage of the target nucleic acid.
[Claim 38]
The method of any one of claims 18-37, wherein the modifying comprises introducing a mutation at the target locus selected from an insertion, a deletion, and a frameshift mutation.
[Claim 39]
The method of any one of claims 18-38, further comprising contacting the target nucleic acid with a donor DNA comprising a sequence homologous to the sequence of the target locus under a condition that allows integration of the donor DNA at the target locus.
[Claim 40]
The method of claim 39, wherein the donor DNA is transfected into the cell separately from the guide DNA.
[Claim 41]
The method of claim 40, wherein the donor DNA is transfected into the cell at least about 6 hours after the transfection of the guide DNA.
[Claim 42]
The method of claim 39, wherein the donor DNA and the guide DNA are transfected into the cell simultaneously, and wherein the donor DNA and the guide DNA are separately packed with a transfection agent.
[Claim 43]
The method of any one of claims 39-42, wherein the donor DNA encodes a selection marker.
[Claim 44]
The method of claim 43, further comprising assessing the cell for expression of the selection marker.
[Claim 45]
The method of any one of claims 18-44, wherein the modifying comprises inducing a phenotypic change to the cell.
[Claim 46]
The method of claim 45, further comprising assessing the phenotypic change to the cell.
[Claim 47]
The method of any one of claims 18-46, further comprising sequencing the target nucleic acid after the modifying.
[Claim 48]
The method of any one of claims 18-47, wherein the modifying comprises altering expression of the target nucleic acid.
[Claim 49]
The method of any one of claims 39-48, wherein the modifying comprises introducing a knockout mutation at the target locus.
[Claim 50]
The method of any one of claims 39-48, wherein the modifying comprises knocking in an exogenous sequence at the target locus, wherein the donor DNA comprises the exogenous sequence.
[Claim 51]
The method of any one of claims 39-48, wherein the modifying comprises introducing a substitution mutation at the target locus, wherein the donor DNA comprises the substitution mutation.
[Claim 52]
The method of claim 51, wherein the substitution mutation is a single nucleotide substitution.
[Claim 53]
A kit comprising: (a) a first composition comprising a single-stranded guide DNA and a first transfection agent; and (b) a second composition comprising a nucleic acid encoding an Ago protein and a second transfection agent, wherein the Ago protein and the guide DNA form a complex that is capable of specifically recognizing a target locus at a temperature of about 10℃ to about 60℃, and wherein the target locus comprises a sequence that is complementary to the sequence of the guide DNA.

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