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1. WO2020114893 - CRISPR GUIDE-RNA EXPRESSION STRATEGIES FOR MULTIPLEX GENOME ENGINEERING

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CLAIMS

1. A method for expression within a cell of at least two functional guide-RNA molecules, comprising contacting a cell with a plurality of single-stranded oligonucleotide members and a linear double-stranded polynucleotide member such that these are introduced into the cell, wherein the members of the plurality of single-stranded oligonucleotides are capable of assembly within a cell into a double-stranded polynucleotide encoding an array of at least two functional guide-RNA molecules, wherein each guide-RNA molecule comprises at least a guide sequence and an RNA processing sequence.

2. A method according to claim 1 , wherein the RNA processing sequence is a Cas12a Direct Repeat (DR) sequence, a Csy4 recognition sequence, a self-processing ribozyme or a tRNA.

3. A method according to any of claims 1 and 2, wherein the cell expresses a functional Cas12a-like enzyme, a functional Csy4 and/or a functional Cas9-like enzyme or wherein in the cell a functional Cas12a-like enzyme, a functional Csy4 and/or a functional Cas9-like enzyme is present.

4. A method according to any one of claims 1 to 3, wherein a part at the 5’-end of the double-stranded polynucleotide encoding at least two functional guide-RNA molecules has sequence identity with a part at one terminal part of the linear double-stranded polynucleotide and wherein a part at the 3’-end of the double-stranded polynucleotide encoding the array of at least two functional guide-RNA molecules has sequence identity with the other terminal part of the linear double-stranded polynucleotide, such that the plurality of single-stranded oligonucleotide members, when assembled, can assemble together with the linear double-stranded polynucleotide into the double-stranded polynucleotide construct.

5. A method according to any one of claims 1 to 4, wherein the oligonucleotide members comprise overlapping portions at least 10bases each, such that they are capable of assembly within a cell into a double-stranded polynucleotide encoding an array of at least two functional guide-RNA molecules.

6. A method according to any one of claims 1 to 5, wherein the double-stranded polynucleotide encodes an array of three, four, five, six or more functional guide-RNA molecules.

7. A method according to any one of claims 1 to 6, wherein the plurality of single-stranded oligonucleotide members comprises at least three, four, five, six or more members.

8. A method according to any one of claims 1 to 7, wherein the linear double-stranded polynucleotide is a vector comprising a selectable marker.

9. A method according to any one of claims 1 to 8, wherein the assembly results in a circular double-stranded polynucleotide construct of pre-determined sequence.

10. A method according to any one of claims 1 to 9, wherein the linear double-stranded polynucleotide comprises a promoter, or a part thereof, that, after assembly, is operably linked to the polynucleotide encoding the array of at least two functional guide-RNA molecules.

11. A method according to any one of claims 1 to 10, wherein the linear double-stranded polynucleotide comprises a terminator that, after assembly, is operably linked to the polynucleotide encoding the array of at least two functional guide-RNA molecules.

12. A method according to any one of claims 1 to 11 , wherein the polynucleotide encoding the array of at least two functional guide-RNA molecules comprises a terminator that is operably linked to it.

13. A method according to any one of claims 1 to 12, wherein two reverse oligonucleotide members and one forward oligonucleotide member are used for a functional guide-RNA molecule or wherein two forward oligonucleotide members and one reverse oligonucleotide member are used for a functional guide-RNA molecule.

14. A cell obtainable by or obtained by a method according to any one of claims 1 to 13.

15. A method for the production of a compound of interest comprising, culturing a cell according to claim 14, said cell comprising a polynucleotide encoding a compound of interest, under conditions conducive to the production of the compound of interest, and optionally isolating and/or purifying the compound of interest.