Multiplex RNA-Guided Genome Engineering

• US 20160168592A1
• Filed: 07/08/2014
• Published: 06/16/2016
• Est. Priority Date: 07/09/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Dicarlo, James, Church, George M.
• Granted Patent: US 11,459,585 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

Methods of multiplex genome engineering in cells using Cas9 is provided which includes a cycle of steps of introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells.

Claims

1. A method of making multiple alterations to target DNA in a cell expressing an enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner comprising

(a) introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA,

introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences,

wherein the one or more RNAs and the one or more donor nucleic acid sequences are expressed,

wherein the one or more RNAs and the enzyme co-localize to the target DNA, the enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the cell, and

repeating step (a) multiple times to produce multiple alterations to the DNA in the cell.

2. The method of claim 1 wherein the enzyme is an RNA-guided DNA binding protein.

3. The method of claim 1 wherein the enzyme is Cas9.

4. The method of claim 1 wherein the cell is a eukaryotic cell.

5. The method of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.

6. The method of claim 1 wherein the RNA is between about 10 to about 500 nucleotides.

7. The method of claim 1 wherein the RNA is between about 20 to about 100 nucleotides.

8. The method of claim 1 wherein the one or more RNAs is a guide RNA.

9. The method of claim 1 wherein the one or more RNAs is a tracrRNA-crRNA fusion.

10. The method of claim 1 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

11. The method of claim 1 wherein the one or more donor nucleic acid sequences are inserted by recombination.

12. The method of claim 1 wherein the one or more donor nucleic acid sequences are inserted by homologous recombination.

13. The method of claim 1 wherein the one or more RNAs and the one or more donor nucleic acid sequences are present on one or more plasmids.