CRISPR ENABLED MULTIPLEXED GENOME ENGINEERING

• US 20170240922A1
• Filed: 02/11/2015
• Published: 08/24/2017
• Est. Priority Date: 02/11/2014
• Status: Active Grant

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: GILL, Ryan T., GARST, Andrew
• Granted Patent: US 10,266,849 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1024 In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/902 using homologous recombination
• 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 2320/12 in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Described herein are methods and vectors for rational, multiplexed manipulation of chromosomes within open reading frames (e.g., in protein libraries) or any segment of a chromosome in a cell or population of cells, in which various CRISPR systems are used.

Claims

1-53. -53. (canceled)

54. A synthesized cassette comprising:

(i) at least one editing cassette comprising (a) a region homologous to a target region of a nucleic acid in a cell, (b) a mutation of at least one nucleotide relative to said target region, and (c) a first protospacer adjacent motif (PAM) mutation; and

(ii) a nucleic acid encoding at least a portion of a guide RNA (gRNA) comprising (a) a region complementary to a portion of the target region.

55. The synthesized cassette of claim 54, wherein said nucleic acid encoding at least a portion of a gRNA further comprises (b) a region that interacts with, or recruits, a endonuclease.

56. The synthesized cassette of claim 54, wherein said editing cassette serves as a trans-barcode for subsequent sequencing.

57. The synthesized cassette of claim 54, wherein the target region is within a non-coding region.

58. The synthesized cassette of claim 54, wherein the target region is within a gene of interest within the cell.

59. The synthesized cassette of claim 58, wherein the mutation of at least one nucleotide is within at least one codon of the gene of interest.

60. The synthesized cassette of claim 58, wherein the PAM mutation is outside of a reading frame of the gene of interest.

61. The synthesized cassette of claim 58, wherein the gene of interest is a prokaryotic gene.

62. The synthesized cassette of claim 58, wherein the gene of interest is a eukaryotic gene.

63. The synthesized cassette of claim 54, wherein the mutation of at least one codon is within 100 nucleotides of the PAM mutation.

64. The synthesized cassette of claim 54, synthesized by array-based synthesis.

65. The synthesized cassette of claim 54, synthesized by parallel synthesis.

66. The synthesized cassette of claim 54, wherein said gRNA comprises a single chimeric gRNA.

67. The synthesized cassette of claim 54, wherein said gRNA comprises a crRNA and a trRNA.

68. The synthesized cassette of claim 54, further comprises at least two priming sites.

69. The synthesized cassette of claim 54, wherein the endonuclease is a Cas9 nuclease.

70. The synthesized cassette of claim 54, further comprising a promoter.

71. A synthesized cassette library comprising at least two of the synthetic cassette of claim 54.

72. The synthesized cassette library of claim 71, comprising at least 8,000 different mutations.

73. The synthesized cassette library of claim 71, comprising at least 27,000 different rationally designed oligonucleotides.

74. The synthesized cassette library of claim 71, comprising about 10⁴-10⁶ different synthesized cassettes.

75. The synthesized cassette library of claim 71, comprising at least 10⁴ different synthesized cassettes.

76. The synthesized cassette library of claim 71, comprising at least 10⁵ different synthesized cassettes.

77. A vector comprising a synthesized cassette, wherein the synthesized cassette comprises:

(i) at least one editing cassette comprising (a) a region homologous to a target region of a nucleic acid in a cell, (b) a mutation of at least one nucleotide relative to said target region, and (c) a first protospacer adjacent motif (PAM) mutation; and

(ii) a nucleic acid encoding at least a portion of a guide RNA (gRNA) comprising (a) a region complementary to a portion of the target region.

78. The vector of claim 77, further comprising a nucleic acid encoding a portion of a gRNA comprising (b) a region that interacts with, or recruits, an endonuclease, thereby generating a functional gRNA comprising (b) and (a) a region complementary to a portion of the target region.

79. The vector of claim 77, further comprising a selectable marker.

80. The vector of claim 77, further comprising a nucleic acid encoding said endonuclease.

81. A method of genome engineering comprising:

(a) introducing a vector from claim 77 into a first population of cells, thereby producing a second population of cells comprising said vector;

(b) maintaining said second population of cells under conditions in which Cas9 nuclease is expressed, wherein said Cas9 nuclease is encoded on said vector, encoded on a second vector, or encoded on genomic DNA of said second population of cells, thereby producing a third population of cells comprising said vector and said target region comprising said PAM mutation;

(c) culturing the product of (b) under conditions appropriate for cell viability, thereby producing viable cells comprising a destination library;

(d) obtaining viable cells produced in (c); and

(e) identifying said mutation of at least one nucleotide by sequencing.

82. A method of generating a vector library comprising,

providing a plurality of synthesized editing oligonucleotides, each individually comprising (a) a region homologous to a target region of a nucleic acid in a cell, (b) a mutation of at least one nucleotide relative to said target region, and (c) a temporary protospacer adjacent motif (PAM) mutation,

transforming said plurality of synthesized editing oligonucleotides and a first guide RNA into a population of cells comprising said target region,

maintaining said cells under conditions in which Cas9 nuclease is expressed, wherein the Cas9 nuclease is encoded on genomic DNA within the cell or encoded on a polynucleotide which is transformed into the cells,

enriching for cells comprising said temporary PAM mutation, thereby generating a donor library comprising target regions comprising said mutation in at least one nucleotide and said temporary PAM mutation,

amplifying said donor library to incorporate a first PAM mutation, thereby generating an amplified donor library comprising polynucleotides comprising a mutated target region and said first PAM mutation,

incorporating a nucleic acid encoding at least one guide RNA and a promoter into the polynucleotides of said donor library, thereby generating said vector library.

83. A cell comprising:

(a) a nucleic acid comprising a target region,

(b) a vector comprising

(i) at least one editing cassette comprising (a) a region homologous to the target region of a nucleic acid in a cell, (b) a mutation of at least one nucleotide relative to said target region, and (c) a first protospacer adjacent motif (PAM) mutation; and

(ii) a nucleic acid encoding at least one guide RNA (gRNA) comprising (a) a region complementary to a portion of the target region; and

(b) a region that interacts with a Cas9 nuclease, and

(c) a Cas9 nuclease.