Various knockouts of heterozygous ELANE gene alleles using guide sequences of 21-30 nucleotides in length.

Abstract

This invention provides a method for knocking out the expression of a dominant mutant allele by disrupting the dominant mutant allele or by degrading the resulting mRNA. [Solution] A method for inactivating a mutant allele of the neutrophil elastase gene (ELANE gene) having a mutation related to severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) in a cell, wherein the cell is a specific heterozygote expressing an allele having a mutation encoding a disease-causing mutant protein, and the method comprises introducing into the cell a composition comprising a CRISPR nuclease or a sequence encoding the CRISPR nuclease and a first RNA molecule comprising a guide sequence of 21 to 30 nucleotides, wherein the complex of the CRISPR nuclease and the first RNA molecule acts to break the double-strand of the mutant allele of the ELANE gene.

Claims

1. A method for inactivating mutant alleles of the neutrophil elastase gene (ELANE gene) having mutations related to severe congenital neutropenia (SCN) or cyclic neutropenia (CyN) within cells, wherein the cells are rs1683564, rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, r The polymorphic region is heterojunction in one or more polymorphic regions selected from the group consisting of s781452480, rs371057361, rs570466264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs71335276 and rs8107095, and the method is, CRISPR nuclease or a sequence encoding the CRISPR nuclease, The first RNA molecule containing a guide sequence region of 21-30 nucleotides. The process involves introducing a composition containing the above into the cells, Herein, the complex of the CRISPR nuclease and the first RNA molecule acts to break the double-strand allele of the ELANE gene.

2. The method according to claim 1, wherein the guide sequence portion of the first RNA molecule comprises 21 to 30 consecutive nucleotides including the nucleotide sequence shown in any one of SEQ ID NOs: 2517, 2601, 2464-2516, 2518-2600, 2602-2613, 1-2463, 2614-3751, or 1-2953.

3. The method according to claim 1, wherein the cells are heterozygous in one or more polymorphic sites selected from the group consisting of rs1683564, rs9749274, rs740021, rs199720952, rs28591229, rs71335276, rs3826946, rs10413889, rs3761005, rs10409474, rs3761007, rs17216649, rs10469327, rs8107095, rs10414837 and rs10424470.

4. The method according to any one of claims 1 to 3, wherein the polymorphic region is rs1683564, and the guide sequence portion of the first RNA molecule comprises 21 to 30 consecutive nucleotides including the nucleotide sequence shown in any one of SEQ ID NOs: 2517, 2601, 2464 to 2516, 2518 to 2600, 2602 to 2613, 1 to 2463, and 2614 to 3751.

5. The method according to any one of claims 1 to 4, wherein the complex of the CRISPR nuclease and the first RNA molecule acts on a double-strand break of the mutant allele of the ELANE gene, and the mutant allele becomes a target for the double-strand break based on one or more polymorphic sites.

6. The method according to any one of claims 1 to 5, further comprising introducing a second RNA molecule containing a guide sequence portion capable of forming a complex with a CRISPR nuclease, wherein the complex of the second RNA molecule and the CRISPR nuclease acts on a second double-strand break of the ELANE gene.

7. The method according to claim 6, wherein the second double-strand break occurs within the non-coding region of the ELANE gene.

8. The method according to claim 7, wherein the non-coding region of the ELANE gene is intron 4.

9. The method according to any one of claims 6 to 8, wherein the guide sequence portion of the second RNA molecule comprises 21 to 30 consecutive nucleotides including the nucleotide sequence shown in any one of sequence numbers 2954 to 3751.

10. The method according to any one of claims 6 to 9, wherein the cell is heterozygous at rs10414837 or rs3761005, and the complex of the second RNA molecule and CRISPR nuclease acts on a double-strand break at intron 4 of the ELANE gene.

11. The method according to any one of claims 6 to 9, wherein the cells are heterozygous at rs1683564, and the complex of the second RNA molecule and CRISPR nuclease acts to break the double strand of intron 4 of the ELANE gene.

12. The method according to any one of claims 6 to 11, wherein the second RNA molecule includes a guide sequence portion of 21 to 30 nucleotides.

13. The procedure includes collecting cells containing mutations in the ELANE gene related to SCN or CyN from subjects who have mutations in the ELANE gene related to SCN or CyN, and / or who are suffering from SCN or CyN, the subjects being rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs3 The method according to any one of claims 1 to 12, wherein one or more polymorphic sites selected from the group consisting of 71057361, rs570466264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276 and rs8107095 are heterojunct.

14. First, select subjects who have mutations in the ELANE gene related to SCN or CyN, and / or who are suffering from SCN or CyN. These subjects include rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs The method according to claim 13, wherein the subject is heterozygous in one or more polymorphic sites selected from the group consisting of 1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276 and rs8107095, and comprises collecting the cells from the subject.

15. The method according to claim 13 or 14, comprising collecting the cells from the subject by mobilization and / or apheresis.

16. The method according to claim 15, comprising collecting the cells from the subject by bone marrow aspiration.

17. The method according to any one of claims 1 to 16, wherein the cells are stimulated before the composition is introduced into the cells.

18. The method according to any one of claims 13 to 17, further comprising culturing the cells.

19. The method according to claim 18, wherein the cells are cultured together with one or more stem cell factors (SCF), IL-3, and GM-CSF.

20. The method according to claim 18 or 19, wherein the cells are cultured together with at least one cytokine.

21. The method according to claim 20, wherein the at least one cytokine is a recombinant human cytokine.

22. The method according to any one of claims 1 to 21, wherein the cell is one of a plurality of cells, and the composition comprising the first RNA molecule or the first RNA molecule and the second RNA molecule is introduced into at least the cell and another cell of the plurality of cells, and the mutant allele of the ELANE gene is inactivated in at least the cell and another cell of the plurality of cells, thereby obtaining a plurality of modified cells.

23. The method according to any one of claims 1 to 22, wherein introducing the composition containing the first RNA molecule or introducing the second RNA molecule comprises electroporation of the cell.

24. Modified cells obtained by the method described in claim 22 or 23.

25. Modified cells obtained by culturing the cells described in claim 24.

26. The modified cell according to claim 24 or 25, wherein the cell is capable of engraftment.

27. A modified cell according to any one of claims 24 to 26, which can produce progeny cells.

28. The modified cell according to claim 27, which can produce offspring cells after engraftment.

29. The modified cell according to claim 28, which can produce offspring cells after self-engraftment.

30. The modified cell according to claim 28 or 29, which can produce progeny cells for at least 12 months or at least 24 months after engraftment.

31. The modified cell according to any one of claims 24 to 30, wherein the modified cell is a hematopoietic stem cell and / or progenitor cell (HSPC).

32. The modified cells are CD34 ＋ The modified cell according to claim 31, which is a hematopoietic stem cell.

33. The modified cells according to any one of claims 24 to 32, wherein the modified cells are bone marrow cells or peripheral blood mononuclear cells (PMCs).

34. Modified cells in which at least a portion of one allele of the ELANE gene is missing.

35. The modified cells mentioned above are rs7250194, rs397773837, rs759823713, rs12976041, rs55921706, rs2007647, rs7255385, rs351108, rs6510983, rs375312008, rs1234492733, rs8111201, rs3761010, rs11881698, rs17223066, rs74176357, and rs20198487. The modified cell according to claim 34, which is modified from a cell that is heterozygous in one or more polymorphic sites selected from the group consisting of 0, rs141213775, rs111361200, rs3761001, rs55793725, rs55791968, rs953484068, rs56234325, rs4807932, rs9304953, rs71335273, rs868711974 and rs570466264.

36. A composition comprising modified cells according to any one of claims 24 to 35 and a pharmaceutically acceptable carrier.

37. A method for producing the composition according to claim 36 in vitro or ex vivo, comprising mixing the cells with the pharmaceutically acceptable carrier.

38. A method for producing a composition containing modified cells in vitro or ex vivo, wherein the method is: (a) Subjects who have an ELANE gene mutation related to SCN or CyN, and / or are suffering from SCN or CyN, and whose names are rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs1041904 The process of isolating HSPCs from cells collected from a subject that is heterozygous in one or more polymorphic sites selected from the group consisting of 080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276 and rs8107095, and collecting the cells from the subject; (b) CRISPR nuclease or a sequence encoding the CRISPR nuclease; and The first RNA molecule containing a guide sequence region of 21-30 nucleotides. A composition containing is introduced into the cells of step (a), A step of obtaining modified cells by inactivating the mutant allele of the ELANE gene in one or more cells. Here, the complex of the CRISPR nuclease and the first RNA molecule acts to break the double-strand allele of the ELANE gene in one or more cells. In some cases, a second RNA molecule containing a guide sequence portion capable of forming a complex with the CRISPR nuclease is introduced into the cells, and the complex of the second RNA molecule and the CRISPR nuclease acts on a second double-strand break of the ELANE gene in one or more cells; in some cases, (c) A step of culturing the modified cells from step (b), Here, the modified cells are capable of engraftment and produce progeny cells after engraftment. A method that includes this.

39. (a) Subjects who have an ELANE gene mutation related to SCN or CyN and / or are infected with SCN or CyN, and whose names are rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466264, rs A step of isolating HSPCs from cells collected from subjects that are heterozygous in one or more polymorphic sites selected from the group consisting of 1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276, and rs8107095; (b) CRISPR nuclease or a sequence encoding the CRISPR nuclease; and The first RNA molecule containing a guide sequence region of 21-30 nucleotides. A composition containing is introduced into the cells of step (a), A step of obtaining modified cells by inactivating the mutant allele of the ELANE gene in one or more cells. Here, the complex of the CRISPR nuclease and the first RNA molecule acts to break the double-strand allele of the ELANE gene in one or more cells. In some cases, a second RNA molecule containing a guide sequence portion capable of forming a complex with the CRISPR nuclease is introduced into the cells, and the complex of the second RNA molecule and the CRISPR nuclease acts on a second double-strand break of the ELANE gene in one or more cells; in some cases, (c) A step of culturing the cells of step (b), wherein the modified cells are capable of engraftment and produce progeny cells after engraftment; and, (d) A step of administering the cells of step (b) or step (c) to the subject, Use of a composition prepared in vitro by a method comprising the above, for the treatment of SCN or CyN in the subject.

40. A method for treating a subject suffering from SCN or CyN, comprising administering a therapeutically effective amount of a modified cell according to any one of claims 24 to 34, the composition according to claim 35, or a composition prepared by the method according to claim 36 or 37.

41. A method for treating SCN or CyN in subjects having ELANE gene mutations related to SCN or CyN, wherein the subjects are rs10424470, rs4807932, rs10414837, rs376107533, rs3761010, rs351108, rs3826946, rs10413889, rs3761007, rs10409474, rs3761005, rs351107, rs3761001, rs740021, rs781452480, rs371057361, rs570466 The polymorphic region is heterojunction in one or more polymorphic regions selected from the group consisting of 264, rs1041904080, rs7250194, rs17216649, rs199720952, rs6510983, rs17223066, rs7255385, rs9749274, rs111361200, rs112639467, rs141213775, rs28591229, rs10469327, rs3834645, rs1683564, rs71335276 and rs8107095, and the method is, (a) A step of isolating HSPCs from cells collected from the subject; (b) CRISPR nuclease or a sequence encoding the CRISPR nuclease; and The first RNA molecule containing a guide sequence region of 21-30 nucleotides. A composition containing is introduced into the cells of step (a), A step of obtaining modified cells by inactivating the mutant allele of the ELANE gene in one or more cells. Here, the complex of the CRISPR nuclease and the first RNA molecule acts to break the double-strand allele of the ELANE gene in one or more cells. In some cases, a second RNA molecule containing a guide sequence portion capable of forming a complex with the CRISPR nuclease is introduced into the cells, and the complex of the second RNA molecule and the CRISPR nuclease acts on a second double-strand break of the ELANE gene in one or more cells; in some cases, (c) A step of culturing the cells of step (b), wherein the modified cells are capable of engraftment and produce progeny cells after engraftment; and, (d) A step of administering the cells of step (b) or step (c) to the subject, A method comprising treating SCN or CyN in the subject.

42. A method for treating SCN or CyN in subjects having ELANE gene mutations related to SCN or CyN, wherein the subjects are rs7250194, rs397773837, rs759823713, rs12976041, rs55921706, rs2007647, rs7255385, rs351108, rs6510983, rs375312008, rs1234492733, rs8111201, rs3761010, rs11881698, r The polymorphic region is heterozygous in one or more polymorphic regions selected from the group consisting of s17223066, rs74176357, rs201984870, rs141213775, rs111361200, rs3761001, rs55793725, rs55791968, rs953484068, rs56234325, rs4807932, rs9304953, rs71335273, rs868711974 and rs570466264, and the method is, The procedure includes administering the subject's own modified cells or offspring of the subject's own modified cells, wherein the self-modified cells are modified to produce double-strand breaks in the mutant allele of the ELANE gene. Here, the double-strand break is caused by introducing a composition comprising a CRISPR nuclease or a sequence encoding the CRISPR nuclease and a first RNA molecule into the cell, and the complex of the CRISPR nuclease and the first RNA molecule acts on the double-strand break of the mutant allele of the ELANE gene, thereby inactivating the mutant allele of the ELANE gene within the cell. A method for treating SCN or CyN in the subject as described above.

43. A method for selecting subjects for treatment from a group of subjects diagnosed with SCN or CyN, (a) A step of obtaining cells from each of the aforementioned group of subjects; (b) A step of screening each target cell for mutations in the ELANE gene related to SCN or CyN, and selecting only those cells that have mutations in the ELANE gene related to SCN or CyN; (c) A step of screening the target cells selected in step (b) by sequencing for heterozygosity at one or more polymorphic sites selected from the group consisting of rs10414837, rs3761005, and rs1683564; (d) A step of selecting only subjects who have heterozygous cells in one or more polymorphic sites for treatment; (e) A step of obtaining hematopoietic stem and progenitor cells (HSPCs) from the bone marrow of the subject by either aspiration or mobilization of peripheral blood and apheresis; (f) One or more CRISPR nucleases or sequences encoding one or more CRISPR nucleases A first RNA molecule comprising a guide sequence portion having 21 to 30 consecutive nucleotides, including the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 2953, which targets the nucleotide bases of the heterozygous alleles of one or more polymorphic regions present on the mutant allele of the ELANE gene, and A second RNA molecule containing a guide sequence portion that targets a sequence in intron 4 of the ELANE gene, wherein the guide sequence portion of the second RNA molecule has 21 to 30 consecutive nucleotides including the nucleotide sequence shown in any one of sequence numbers 2954 to 3751. The process of introducing it into the HSPC of process (e); Here, the complex of the first RNA molecule and CRISPR nuclease acts on the first double-strand break of the mutant allele of the ELANE gene in one or more HSPC cells, and the complex of the second RNA molecule and CRISPR nuclease acts on the second double-strand break of intron 4 of the two alleles of the ELANE gene in the one or more HSPC cells in which the complex of the first RNA molecule and CRISPR nuclease acted on the first double-strand break, thereby obtaining modified cells. (g) A step of administering the modified cells of step (f) to the subject; Includes, A method for treating the SCN or CyN of the aforementioned target.

44. An RNA molecule containing a guide sequence region having 21 to 30 consecutive nucleotides, including the nucleotide sequence shown in any one of sequence numbers 1 to 3751.

45. A composition comprising the RNA molecule described in claim 44 and a second RNA molecule including a guide sequence portion, wherein the guide sequence portion of the second RNA molecule optionally has 21 to 30 consecutive nucleotides including the nucleotide sequence shown in any one of SEQ ID NOs: 2954 to 3751.

46. The composition according to claim 45, wherein the second RNA molecule targets the non-coding region of the ELANE gene.

47. The composition according to claim 45 or 46, wherein the nucleotide sequence of the guide sequence portion of the second RNA molecule is different from the nucleotide sequence of the guide sequence portion of the first RNA molecule.

48. The composition according to any one of claims 45 to 47, wherein the first RNA molecule and / or the second RNA molecule further comprises a portion having a sequence that binds to a CRISPR nuclease.

49. The composition according to claim 48, wherein the sequence that binds to the CRISPR nuclease is a tracrRNA sequence.

50. The composition according to any one of claims 45 to 49, wherein the first RNA molecule and / or the second RNA molecule further comprises a portion having a tracr-mate sequence.

51. The composition according to any one of claims 45 to 50, wherein the second RNA molecule further comprises one or more linkers.

52. The composition according to any one of claims 45 to 51, wherein the length of the first RNA molecule and / or the second RNA molecule is at most 300 nucleotides.

53. The composition according to any one of claims 45 to 52, further comprising one or more CRISPR nucleases or sequences encoding the one or more CRISPR nucleases, and / or one or more tracrRNA molecules or sequences encoding the one or more tracrRNA molecules.

54. A method for inactivating a mutant ELANE allele in a cell, comprising delivering the RNA molecule described in claim 44 or the composition described in any one of claims 43 to 51 to the cell.

55. A method for treating SCN or CyN, comprising delivering to a subject suffering from SCN or CyN the RNA molecule described in claim 44, the composition described in any one of claims 43 to 51, cells modified with the RNA molecule described in claim 44, or the composition described in any one of claims 43 to 51.

56. The method according to claim 54 or 55, wherein the one or more CRISPR nucleases and / or the tracrRNA and RNA molecules are substantially delivered to the target and / or the cells simultaneously or at different times.

57. The aforementioned method, (a) Deleting an exon containing the disease-causing mutation from a mutant allele, wherein the first RNA molecule or the first RNA molecule and the second RNA molecule target a portion of the exon or a region adjacent to the exon; (b) Deleting multiple exons, entire open reading frames of a gene, or an entire gene; (c) The first RNA molecule or the first RNA molecule and the second RNA molecule target a signal sequence that is alternatively spliced ​​between the exons and introns of the mutant allele; (d) The second RNA molecule targets sequences present in both the mutant allele and the functional allele; (e) The second RNA molecule targets an intron; or (f) Inserting or deleting the mutant allele by the error-prone non-homologous end joining (NHEJ) mechanism, thereby causing a frameshift in the sequence of the mutant allele. (g) In some cases, the frameshift may inactivate or knock out the mutant allele. (h) Preferably, the frameshift forms an early stop codon in the mutant allele, or the frameshift results in nonsense codon-mediated mRNA degradation of the transcript of the mutant allele. The method according to any one of claims 54 to 56, including the method described in any one of claims 54 to 56.

58. The method according to any one of claims 54 to 57, wherein the inactivation or treatment yields a truncated protein encoded by the mutant allele and a functional protein encoded by the functional allele.

59. (a) The cell or subject is heterozygous in rs10414837 or rs3761005, and the complex of the second RNA molecule and the CRISPR nuclease acts on the double-strand break of intron 4 of the ELANE gene, or (b) The method according to any one of claims 54 to 58, wherein the cell or subject is heterozygous in rs1683564, and the complex of the second RNA molecule and the CRISPR nuclease acts to break the double strand of intron 4 of the ELANE gene.

60. Use of the RNA molecule described in claim 44, the composition described in any one of claims 36, 45 to 53, or a composition produced by the method described in claim 37 or 38, for the purpose of inactivating a mutant ELANE allele in a cell.

61. A pharmaceutical for use inactivating a mutant ELANE allele in a cell, comprising the RNA molecule described in claim 44, the composition described in any one of claims 36, 45 to 53, or a composition produced by the method of claim 37 or 38, wherein the pharmaceutical is delivered to the cell by the RNA molecule described in claim 44, the composition described in any one of claims 36, 45 to 53, or a composition produced by the method of claim 37 or 38.

62. Use of the method according to any one of claims 1 to 23, the modified cells according to any one of claims 24 to 35, the composition according to any one of claims 36, 45 to 53, the composition produced by the method according to claim 37 or 38, or the RNA molecule according to claim 44, for the treatment, mitigation, or prevention of SCN or CyN in subjects who are affected by or at risk of being affected by SCN or CyN.

63. A pharmaceutical for use in the treatment, alleviation, or prevention of SCN or CyN, comprising the RNA molecule described in claim 44, the composition described in any one of claims 36, 45 to 53, the composition produced by the method described in claim 37 or 38, or the modified cells described in any one of claims 24 to 35, wherein the pharmaceutical is delivered to a subject who is suffering from or at risk of suffering from SCN or CyN, the RNA molecule described in claim 44, the composition described in any one of claims 36, 45 to 53, the composition produced by the method described in claim 37 or 38, or the modified cells described in any one of claims 24 to 35.

64. A kit for inactivating a mutant ELANE allele in a cell, comprising the RNA molecule, CRISPR nuclease or a sequence encoding the CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding the tracrRNA; and instructions for delivering the RNA molecule, wherein the CRISPR nuclease or the sequence encoding the CRISPR nuclease, and / or a tracrRNA molecule or a sequence encoding the tracrRNA are directed to the cell for inactivating the mutant ELANE allele in the cell.

65. A kit for treating a target SCN or CyN, comprising the RNA molecule, CRISPR nuclease or sequence encoding the CRISPR nuclease, and / or tracrRNA molecule or sequence encoding the tracrRNA; and instructions for delivering the RNA molecule, wherein the CRISPR nuclease or sequence encoding the CRISPR nuclease, and / or tracrRNA molecule or sequence encoding the tracrRNA are directed to a subject who has or is at risk of having SCN or CyN for the treatment of SCN or CyN.

66. A kit for inactivating a mutant ELANE allele in a cell, comprising: a composition according to any one of claims 36, 45-53; a composition prepared by the method described in claim 37 or 38; or a modified cell according to any one of claims 24-35; and instructions for delivering the composition to the cell in order to inactivate the ELANE gene in the cell.

67. A kit for treating a target SCN or CyN, comprising a composition according to any one of claims 36, 45-53, a composition manufactured by the method described in claim 37 or 38, or modified cells according to any one of claims 24-35, and instructions for delivering the composition according to any one of claims 36, 45-53, wherein the composition manufactured by the method described in claim 37 or 38 or the modified cells according to any one of claims 24-35 is directed to a subject who has or is at risk of developing SCN or CyN for the treatment of SCN or CyN.

68. The method according to any one of claims 1 to 23, 38, 41, 42, and 43, or the kit according to claim 64 or 65, wherein the CRISPR nuclease exhibits cleavage activity when used with an RNA molecule containing a guide sequence portion of 21 to 30 nucleotides.

69. The aforementioned CRISPR nuclease is effective against Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces roseum, and Streptosporangium. Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonyi watsonii), Cyanothece sp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difjicile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Francisella cf. novicida Fx1, Alicyclobacillus acidoterrestris, Oleiphilus sp., Bacterium CG09_39_24 The method or kit according to claim 68, derived from either CG09_39_24) or Deltaproteobacteria bacterium.

70. The aforementioned CRISPR nuclease has the following characteristics: (a) The cleavage activity is greater when used with RNA molecules containing a guide sequence of 21 to 23 nucleotides compared to when used with RNA molecules containing a guide sequence of 20 nucleotides or less and / or 24 nucleotides or more; (b) The cleavage activity is greater when used with RNA molecules containing a guide sequence of 21-22 nucleotides compared to when used with RNA molecules containing a guide sequence of 20 nucleotides or less and / or 23 nucleotides or more; and (c) The cleavage activity is maximized when used with an RNA molecule containing a 22-nucleotide guide sequence. A method according to any one of claims 1 to 23, 38, 41 and 42, or a kit according to claim 64 or 65, having one or more of the above.

71. The method or kit according to claim 70, wherein the CRISPR nuclease has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 3789, or the sequence encoding the CRISPR nuclease has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3790 or SEQ ID NO: 3791.

72. The composition containing the aforementioned CRISPR nuclease has the following characteristics: (a) The composition is present in an aqueous solution; (b) The pH of the composition is 6 to 8; (c) The composition does not contain RNase. A method according to any one of claims 1 to 23, a composition according to any one of claims 45 to 53, or a kit according to any one of claims 64 to 67, having one or more of the above.

73. The method according to any one of claims 1 to 23, wherein the first RNA molecule includes a guide sequence portion of 21 to 22 nucleotides.

74. The RNA molecule according to claim 44, wherein the guide sequence portion has 21 to 22 consecutive nucleotides including the nucleotide sequence shown in any one of sequence numbers 1 to 3751.

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