CRISPR enabled multiplexed genome engineering

Patent metadata

Field	Value
Publication number	US-10731180-B2
Application number	US-202016773712-A
Country	US
Kind code	B2
Filing date	Jan 27, 2020
Priority date	Feb 11, 2014
Publication date	Aug 4, 2020
Grant date	Aug 4, 2020

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.

First claim

What is claimed is:

1. A method of generating a cell having a genome edit in one or more target regions, said method comprising:
(a) introducing into said cell an editing cassette, wherein said editing cassette comprises a sequence having the following covalently-linked components:
(i) a nucleic add encoding a gRNA sequence targeting a target region,
(ii) a region homologous to said target region comprising a change in sequence relative to said target region, and
(iii) a mutation preventing nuclease binding at a protospacer adjacent motif (PAM); and

(b) creating the genome edits by introducing into said cell a nuclease compatible with said gRNA sequence; thereby generating the cell having the genome edit.

2. The method of claim 1 , wherein said gRNA sequence is a chimeric guide RNA.

3. The method of claim 1 , wherein said gRNA sequence comprises a crRNA and a tracrRNA.

4. The method of claim 1 , wherein said cell is a prokaryotic cell.

5. The method of claim 1 , wherein said cell is an eukaryotic cell.

6. The method of claim 1 , wherein the change in sequence relative to the target region is within a coding region of the target region.

7. The method of claim 1 , where in the change in sequence relative to the target region is within a non-coding region of the target region.

8. The method of claim 1 , wherein said cell comprises a change in sequence in at least two different target regions.

9. The method of claim 1 , wherein said cell comprises different changes within a same target region.

10. A method of generating a library of cells with genome edits, said method comprising:
(a) obtaining a library of editing cassettes, wherein said library of editing cassette comprises one or more editing cassettes comprising the following, covalently-linked components:
(i) a nucleic acid encoding a gRNA sequence targeting a target region,
(ii) a change in a sequence relative to said target region, and
(iii) a mutation preventing nuclease binding at a protospacer adjacent motif (PAM);

(b) introducing said one or more editing cassettes from said library of editing cassettes into a population of cells,
(c) introducing a nuclease compatible with said gRNA sequence into said population of cells to produce a population of edited cells, and
(d) growing said population of edited cells, thereby generating said library of cells with genome edits.

11. The method of claim 10 , wherein the target region is in a non-coding region.

12. The method of claim 10 , wherein the target region is in a coding region.

13. The method of claim 10 , wherein the library comprises cells with a change in sequence in at least two different target regions.

14. The method of claim 10 , wherein the library comprises cells with at least two different changes within the same target region.

15. The method of claim 10 , wherein said region homologous to said target region is 100 to 120 nucleotides in length.

16. The method of claim 10 , wherein said PAM mutation is 17 to 59 base pairs from said change in sequence relative to said target region.

17. The method of claim 10 , wherein said mutation preventing nuclease binding at a protospaced adjacent motif (PAM) is within a coding region of the target region, and the mutation is a synonymous mutation.

18. The method of claim 10 , wherein said gRNA sequence comprises a crRNA and a tracrRNA.

Assignees

• Univ Colorado Regents

Inventors

• Garst Andrew
• Gill Ryan T

Classifications

• C12N2330/31

  Libraries, arrays · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title