METHODS, SYSTEMS, AND APPARATUS FOR IDENTIFYING TARGET SEQUENCES FOR CAS ENZYMES OR CRISPR-CAS SYSTEMS FOR TARGET SEQUENCES AND CONVEYING RESULTS THEREOF

• US 20190032052A1
• Filed: 06/19/2018
• Published: 01/31/2019
• Est. Priority Date: 12/12/2012
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: ZHANG, Feng, HABIB, Naomi
• Application Number:
  US16/012,692
• Publication Number:
  US 20190032052A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 20/30 Detection of binding sites or motifs
• CPC Class Codes: G16B 20/50 Mutagenesis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Disclosed are locational or positional methods concerning CRISPR-Cas systems, and apparatus therefor.

Claims

1-24. -24. (canceled)

25. A method of identifying one or more unique target sequences for expressing a guide RNA polynucleotide sequence, whereby the one or more unique target sequences are susceptible to being recognized by a CRISPR-Cas system in a genome of a eukaryotic organism, wherein the method comprises:

locating a CRISPR motif;

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome;

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site; and

expressing the guide RNA polynucleotide sequence that recognizes the unique target site in a eukaryotic cell.

26. The method of claim 25, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

27. The method of claim 25, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

28. The method of claim 25, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

29. The method of claim 25, wherein the CRISPR motif is recognized by a Cas9 enzyme.

30. The method of claim 25, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

31. The method of claim 25, wherein the CRISPR motif is NGG.

32. The method of claim 25, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

33. A computer-readable medium comprising codes that, upon execution by one or more processors, implements a method of identifying one or more unique target sequences in a genome of a eukaryotic organism for making a guide RNA polynucleotide sequence, whereby the one or more unique target sequences are susceptible to being recognized by a CRISPR-Cas system, wherein the method comprises:

locating a CRISPR motif;

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome;

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site; and

synthesizing the guide RNA polynucleotide sequence that recognizes the unique target site.

34. The computer-readable medium of claim 33, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

35. The computer-readable medium of claim 33, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

36. The computer-readable medium of claim 33, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

37. The computer-readable medium of claim 33, wherein the CRISPR motif is recognized by a Cas9 enzyme.

38. The computer-readable medium of claim 33, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

39. The computer-readable medium of claim 33, wherein the CRISPR motif is NGG.

40. The computer-readable medium of claim 33, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

41. A computer system for identifying one or more unique target sequences in a genome of a eukaryotic organism for making a guide RNA polynucleotide sequence, the system comprising:

a. a memory unit configured to receive and/or store sequence information of the genome; and

b. one or more processors alone or in combination programmed to (i) locate a CRISPR motif, (ii) analyze a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome, (iii) select the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site and (iv) display the one or more unique target sequences, whereby the one or more unique target sequences is used to make a guide RNA polynucleotide sequence.

42. The system claim 41, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

43. The system of claim 41, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

44. The system of claim 41, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

45. The system of claim 41, wherein the CRISPR motif is recognized by a Cas9 enzyme.

46. The system of claim 41, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

47. The system of claim 41, wherein the CRISPR motif is NGG.

48. The system of claim 41, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

49. A Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a nucleotide sequence encoding a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence,

wherein the polynucleotide sequence comprises:

(a) a guide sequence capable of hybridizing to a unique target sequence in a eukaryotic cell, whereby the unique target sequence is susceptible to being recognized by a CRISPR-Cas system in a genome of a eukaryotic organism, wherein the unique target sequence is identified by a method comprising;

locating a CRISPR motif,

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome,

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site,

(b) a trans-activating CRISPR RNA (tracr) mate sequence, and

(c) a tracrRNA sequence,

wherein (a), (b) and (c) are arranged in a 5′

to 3′

orientation,

wherein the tracrRNA sequence is 50 or more nucleotides in length, and

II. a second regulatory element operably linked to a nucleotide sequence encoding a Type-II Cas9 protein comprising one or more nuclear localization sequences, of sufficient strength to drive accumulation of said Cas9 protein in a detectable amount in the nucleus of a eukaryotic cell;

wherein components I and II are located on the same or different vectors of the system; and

wherein when the nucleotide sequences are transcribed;

the chiRNA assembles into and complexes with the Type II Cas9 protein,

the tracr mate sequence hybridizes to the tracrRNA sequence and

the guide sequence directs sequence-specific binding to the unique target sequence in the eukaryotic cell,

whereby there is formed a CRISPR complex comprising the Type II Cas9 protein complexed with (1) the guide sequence that is hybridized to the unique target sequence in the eukaryotic cell, and (2) the tracr mate sequence that is hybridized to the tracrRNA sequence.

50. The vector system of claim 49, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

51. The vector system of claim 49, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

52. The vector system of claim 49, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

53. The vector system of claim 49, wherein the CRISPR motif is recognized by a Cas9 enzyme.

54. The vector system of claim 49, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

55. The vector system of claim 49, wherein the CRISPR motif is NGG.

56. The vector system of claim 49, wherein the guide RNA sequence is of between 10-30 nucleotides in length.

57. The vector system of claim 49, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).

58. An engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:

I. a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA polynucleotide sequence capable of hybridizing to a unique target sequence of a DNA molecule in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product, and wherein the unique target sequence is susceptible to being recognized by a CRISPR-Cas system in a genome of a eukaryotic organism, and wherein the unique target sequence is identified by a method comprising;

locating a CRISPR motif,

analyzing a sequence upstream of the CRISPR motif to determine if the sequence occurs elsewhere in the genome,

selecting the sequence if it does not occur elsewhere in the genome, thereby identifying a unique target site, and

II. a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,

wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA polynucleotide sequence targets and hybridizes with the unique target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered; and

, wherein the Cas9 protein and the guide RNA do not naturally occur together.

59. The CRISPR-Cas system of claim 58, wherein the sequence upstream of the CRISPR motif is at least 20 bp in length.

60. The CRISPR-Cas system of claim 58, wherein the sequence upstream of the CRISPR motif is at least 12 bp in length.

61. The CRISPR-Cas system of claim 58, wherein the sequence upstream of the CRISPR motif is at least 10 bp in length.

62. The CRISPR-Cas system of claim 58, wherein the CRISPR motif is recognized by a Cas9 enzyme.

63. The CRISPR-Cas system of claim 58, wherein the CRISPR motif is recognized by a SpCas9 enzyme.

64. The CRISPR-Cas system of claim 58, wherein the CRISPR motif is NGG.

65. The CRISPR-Cas system of claim 58, wherein the guide RNA sequence is of between 10-30 nucleotides in length.

66. The CRISPR-Cas system of claim 58, wherein the eukaryotic organism is selected from the group consisting of Homo sapiens (human), Mus musculus (mouse), Rattus norvegicus (rat), Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (roundworm), Sus scrofa (pig) and Bos taurus (cow).