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1. WO2020223514 - NOVEL OMNI-50 CRISPR NUCLEASE

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CLAIMS

What is claimed is:

1. A non-naturally occurring composition comprising a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease.

2. The composition of claim 1, further comprising a DNA-targeting RNA molecule or a DNA polynucleotide encoding a DNA-targeting RNA molecule, wherein the DNA-targeting RNA molecule comprises a nucleotide sequence that is complementary to a sequence in a target region, wherein the DNA-targeting RNA molecule and the CRISPR nuclease do not naturally occur together.

3. The composition of claim 2, wherein the DNA-targeting RNA molecule comprises a crRNA repeat sequence which comprises the sequence GUUUGAGAG.

4. The composition of any one of claims 2 or 3, wherein the DNA-targeting RNA molecule comprises a tracrRNA sequence which comprises one or more sequences selected from SEQ ID NOs: 41-43 and SEQ ID NOs: 149-154.

5. The composition of any one of claims 2-4, wherein the DNA-targeting RNA molecule comprises a nucleotide sequence that can form a complex with the CRISPR nuclease.

6. An engineered, non-naturally occurring composition comprising a CRISPR associated system comprising:

one or more RNA molecules comprising a guide sequence portion linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more RNA molecules; and a CRISPR nuclease comprising an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and

wherein the one or more RNA molecules hybridize to the target sequence, wherein the target sequence is 3' of a Protospacer Adjacent Motif (PAM), and the one or more RNA molecules form a complex with the RNA-guided nuclease.

7. The composition of any one of claims 1-6, further comprising an RNA molecule comprising a nucleotide sequence that can form a complex with a CRISPR nuclease

(tracrRNA) or a DNA polynucleotide comprising a sequence encoding an RNA molecule that can form a complex with the CRISPR nuclease.

8. The composition of any one of claims 2-7, further comprising a donor template for homology directed repair (HDR).

9. The composition of any one of claims 2-8, wherein the composition is capable of editing the target region in the genome of a cell.

10. The composition of any one of claims 1-9, wherein the CRISPR nuclease comprises a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 and the nucleotide sequence that can form a complex with the CRISPR nuclease in the DNA- targeting RNA molecule comprises a sequence selected from SEQ ID NOs: 44-52.

11. A non-naturally occurring composition comprising:

a CRISPR nuclease comprising a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 or a nucleic acid molecule comprising a sequence encoding the CRISPR nuclease; and

one or more RNA molecules, or one or more DNA polynucleotide encoding the one or more RNA molecules, comprising at least one of:

(i) a nuclease-binding RNA nucleotide sequence capable of interacting with/binding to the CRISPR nuclease; and

(ϋ) a DNA-targeting RNA nucleotide sequence comprising a sequence complementary to a sequence in a target DNA sequence,

wherein the CRISPR nuclease is capable of complexing with the one or more RNA molecules to form a complex capable of hybridizing with the target DNA sequence.

12. The composition of claim 11, wherein the CRISPR nuclease and the one or more RNA molecules form a CRISPR complex that is capable of binding to the target DNA sequence to effect cleavage of the target DNA sequence.

13. The composition of any one of claims 11 or 12, wherein the CRISPR nuclease and at least one of the one or more RNA molecules do not naturally occur together.

14. The composition of any one of claims 11-13, wherein:

the CRISPR nuclease comprises an KNA-binding portion and an activity portion that exhibits site-directed enzymatic activity;

DNA-targeting RNA nucleotide sequence comprises a nucleotide sequence that is complementary to a sequence in a target DNA sequence; and

the nuclease-binding RNA nucleotide sequence comprises a sequence that interacts with the RNA-binding portion of the CRISPR nuclease.

15. The composition of any one of claims 11-14, wherein the nuclease-binding RNA nucleotide sequence and the DNA-targeting RNA nucleotide sequence are on a single RNA molecule (sgRNA), wherein the sgRNA molecule can form a complex with the CRISPR nuclease and serve as the DNA targeting module.

16. The non-naturally occurring composition of claim 15, wherein the sgRNA has a length of up to 1000 bases, 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, 100 bases, 50 bases.

17. The composition of any one of claims 1-16, further comprising a donor template for homology directed repair (HDR).

18. The composition of any one of claims 1-17, wherein the CRISPR nuclease has at least 95% identity to the amino acid sequence as set forth in SEQ ID NO: 3 or wherein the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease comprises a sequence of at least a 95% sequence identity to the nucleic acid sequence as set forth in SEQ ID NOs: 11-13.

19. The composition of claim 18, wherein the composition comprises a CRISPR nuclease having at least 95% identity to SEQ ID NO: 3 and an RNA molecule comprising a nucleasebinding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs: 37-45, 87-88, 149-154, and GUUUGAGAG and is suitable to form an active complex with the CRISPR nuclease.

20. The composition of claim 19, wherein the CRISPR nuclease uses a PAM site selected from NGG, NAG, and NGA.

21. The composition of any one of claims 1-20, wherein the CRISPR nuclease comprises at least 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110- 120, 120-130,130-140, or 140-150 amino acid substitutions, deletions, and/or insertions compared to the amino acid sequence of the wild-type of the CRISPR nuclease.

22. The composition of claim 21, wherein the CRISPR nuclease exhibits at least 2%, 5%, 7% 10%, 15%, 20%, 25%, 30, or 35% increased specificity or increased activity compared the wild-type of the CRISPR nuclease, or has altered PAM specificity compared to the wild- type of the CRISPR nuclease.

23. The composition of any one of claims 1-22, wherein the CRISPR nuclease is engineered or non-naturally occurring.

24. The composition of claim 23, wherein the CRISPR nuclease is engineered and comprises unnatural or synthetic amino acids.

25. The composition of any one of claims 23 or 24, wherein the CRISPR nuclease is engineered and comprises one or more of a nuclear localization sequences (NLS), cell penetrating peptide sequences, and/or affinity tags.

26. A method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell the composition of any one of claims 1-25.

27. The method of claim 26, wherein the cell is a eukaryotic cell or a prokaryotic cell.

28. A method of modifying a nucleotide sequence at a target site in the genome of a mammalian cell comprising introducing into the cell (i) a composition comprising a CRISPR nuclease having at least 95% identity to the amino acid sequence of SEQ ID NO: 3 or wherein the nucleic acid molecule comprising a sequence encoding the CRISPR nuclease has at least a 95% nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-13 and (ii) a DNA-targeting RNA molecule, or a DNA polynucleotide encoding a DNA-targeting RNA molecule, comprising a nucleotide sequence that is complementary to a sequence in the target DNA.

29. The method of claim 28, further comprising introducing into the cell: (iii) an RNA molecule comprising a nuclease-binding RNA sequence or a DNA polynucleotide encoding an RNA molecule comprising a nuclease-binding RNA that interacts with the CRISPR nuclease.

30. The method of any one of claims 28 or 29, wherein the DNA targeting RNA molecule is a crRNA molecule suitable to form an active complex with the CRISPR nuclease.

31. The method of any one of claims 28-30, wherein the RNA molecule comprising a nucleasebinding RNA sequence is a tracrRNA molecule suitable to form an active complex with the CRISPR nuclease.

32. The method of any one of claims 38-31, wherein the DNA-targeting RNA molecule and the RNA molecule comprising a nuclease-binding RNA sequence are fused in the form of a single guide RNA molecule.

33. The method of any one of claims 28-32, comprising introducing into the cell: (iv) an RNA molecule comprising a sequence complementaiy to a protospacer sequence.

34. The method of any one of claims 28-33, wherein the CRISPR nuclease forms a complex with the one or more RNA molecules and effects a double strand break in the 3’ of a Protospacer Adjacent Motif (PAM).

35. The method of any one of claims 28-34, wherein the CRISPR nuclease comprises a sequence having at least 95% identity to SEQ ID NO: 3 and an RNA molecule comprising a nuclease-binding RNA nucleotide sequence wherein the nucleotide binding RNA sequence is selected from the group consisting of SEQ ID NOs: 37-45, 87-88, 149-154, and GUUUGAGAG and is suitable to form an active complex with the CRISPR nuclease.

36. The method of claim 35, wherein the CRISPR nuclease uses a PAM site selected from NGG, NAG, and NGA.

37. The method of any one of claims 28-36, wherein the CRISPR nuclease comprises a sequence having at least 95% identity to SEQ ID NO: 3 and uses a PAM site selected from NGG, NAG, and NGA.

38. The composition of any of claims 2-27 or the method of any of claims 28-37 wherein the DNA-targeting RNA molecule comprises a guide sequence portion having 21-22 nucleotides.

39. A non-naturally occurring composition comprising a CRISPR nuclease, wherein the CRISPR nuclease comprises an amino acid sequence corresponding to the amino acid sequence of at least one of Domain A, Domain B, Domain C, Domain D, Domain E, or Domain F of the OMNI-50 nuclease.

40. The composition of claim 39, wherein the CRISPR nuclease comprises a Domain A which comprises at least one of

a) Subdomain A1 having at least 97% sequence identity to amino acids 1 to 50 of SEQ ID NO: 3;

b) Subdomain A2 having at least 97% sequence identity to amino acids 741 to 789 of SEQ ID NO: 3; or

c) Subdomain A3 having at least 97% sequence identity to amino acids 962 to 1096 of SEQ ID NO: 3.

41. The composition of any one of claims 39-40, wherein the CRISPR nuclease comprises a Domain B having at least 97% sequence identity to amino acids 51 to 83 of SEQ ID NO:

3.

42. The composition of any one of claims 39-41, wherein the CRISPR nuclease comprises a Domain C which comprises at least one of

a) Subdomain Cl having at least 97% sequence identity to amino acids 84 to 160 of SEQ ID NO: 3;

b) Subdomain C2 having at least 97% sequence identity to amino acids 161 to 299 of SEQ ID NO: 3; or

c) Subdomain C3 having at least 97% sequence identity to amino acids 300 to 737 of SEQ ID NO: 3.

43. The composition of any one of claims 39-41, wherein the CRISPR nuclease comprises a Domain C which comprises at least one of

a) Subdomain Ca having at least 97% sequence identity to amino acids 84 to 478 of SEQ ID NO: 3; and

b) Subdomain Cb having at least 97% sequence identity to amino acids 479 to 737 of SEQ ID NO: 3.

44. The composition of any one of claims 39-43, wherein Domain C has at least 97% sequence identity to amino acids 84 to 737 of SEQ ID NO: 3.

45. The composition of any one of claims 39-44, wherein the CRISPR nuclease comprises a Domain D having at least 97% sequence identity to amino acids 790 to 961 of SEQ ID NO:

3.

46. The composition of any one of claims 39-45, wherein the CRISPR nuclease comprises a Domain E having at least 97% sequence identity to amino acids 1097 to 1196 of SEQ ID NO: 3.

47. The composition of any one of claims 39-47, wherein the CRISPR nuclease comprises a Domain F having at least 97% sequence identity to amino acids 1197 to 1370 of SEQ ID NO: 3.

48. The composition of claim 39, wherein the CRISPR nuclease comprises Domain A, Domain

B, Domain C, Domain D, Domain E, and Domain F, wherein

a) Domain A comprises

i. Subdomain A1 having at least 97% sequence identity to amino acids 1 to 50 of SEQ ID NO: 3;

ii. Subdomain A2 having at least 97% sequence identity to amino acids 741 to 789 of SEQ ID NO: 3; and

iii. Subdomain A3 having at least 97% sequence identity to amino acids 962 to 1096 of SEQ ID NO: 3;

b) Domain B has at least 97% sequence identity to amino acids 51 to 83 of SEQ ID NO: 3;

c) Domain C has at least 97% sequence identity to amino acids 84 to 737 of SEQ ID NO: 3;

d) Domain D has at least 97% sequence identity to amino acids 790 to 961 of SEQ ID NO: 3;

e) Domain E has at least 97% sequence identity to amino acids 1097 to 1196 of SEQ ID NO: 3; and

f) Domain F has at least 97% sequence identity to amino acids 1197 to 1370 of SEQ ID NO: 3.

49. The composition of any one of claims 39-48, wherein the CRISPR nuclease sequence is at least 100-250, 250-500, 500-1000, or 1000-2000 amino acids in length.

50. A non-naturally occurring composition comprising a peptide, wherein the peptide comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of at least one of Domain A, Domain B, Domain C, Domain D, Domain E, or Domain F of the OMNI-50 nuclease.

51. A non-naturally occurring composition comprising a polynucleotide encoding an amino acid sequence having at least 97% sequence identity to the amino acid sequence of at least one of Domain A, Domain B, Domain C, Domain D, Domain E, or Domain F of the OMNI- 50 nuclease.

52. A non-naturally occurring amino acid sequence having at least 97% sequence identity to the amino acid sequence of at least one of Domain A, Domain B, Domain C, Domain D, Domain E, or Domain F of the OMNI-50 nuclease.

53. A method of modifying a nucleotide sequence at a target site in a cell-free system or the genome of a cell comprising introducing into the cell the composition of any one of claims 39-51.

54. The method of claim 53, wherein the cell is a eukaryotic cell, preferably a mammalian cell or a plant cell.

55. Use of the compositions of any one of claims 39-51 for the treatment of a subject afflicted with a disease associated with a genomic mutation comprising modifying a nucleotide sequence at a target site in the genome of the subject.

56. A method of treating subject having a mutation disorder comprising targeting the composition of any one of claims 39-51 to an allele associated with the mutation disorder.

57. The method of claim 56, wherein the mutation disorder is related to a disease or disorder selected from any of a neoplasia, age-related macular degeneration, schizophrenia, neurological, neurodegenerative, or movement disorder, Fragile X Syndrome, secretase- related disorders, prion-related disorders, ALS, addiction, autism, Alzheimer’s Disease, neutropenia, inflammation-related disorders, Parkinson’s Disease, blood and coagulation diseases and disorders, cell dysregulation and oncology diseases and disorders, inflammation and immune-related diseases and disorders, metabolic, liver, kidney and protein diseases and disorders, muscular and skeletal diseases and disorders, dermatological diseases and disorders, neurological and neuronal diseases and disorders, and ocular diseases and disorders.

58. The method of any one of claims 56 or 57, wherein the mutation disorder is beta thalassemia or sickle cell anemia.

59. The method of claim 58, wherein the allele associated with the disease is BCL11 A.