RNA-Guided Human Genome Engineering

• US 20150232833A1
• Filed: 05/01/2015
• Published: 08/20/2015
• Est. Priority Date: 12/17/2012
• Status: Active Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Mali, Prashant G., Church, George M., Yang, Luhan
• Application Number:
  US14/701,912
• Publication Number:
  US 20150232833A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 2810/55 from bacteria
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.

Claims

1. A method of altering a stem cell comprising

providing to the stem cell a guide RNA sequence complementary to a target nucleic acid sequence of genomic DNA of the stem cell, and

providing to the stem cell a Cas 9 protein that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner.

2. The method of claim 1 wherein the guide RNA is provided to the stem cell by introducing to the stem cell a nucleic acid encoding the guide RNA,

wherein the Cas 9 protein is provided to the stem cell by introducing to the stem cell a nucleic acid encoding the Cas9 protein, and

wherein the stem cell produces the guide RNA and the Cas9 protein, the guide RNA binds to complementary genomic DNA and the Cas 9 protein cleaves the genomic DNA in a site specific manner.

3. The method of claim 1 wherein the stem cell is a human stem cell

4. The method of claim 1 wherein the stem cell is an induced pluripotent stem cell.

5. The method of claim 1 wherein the guide RNA includes between about 10 to about 250 nucleotides.

6. The method of claim 1 wherein the guide RNA includes between about 20 to about 100 nucleotides.

7. The method of claim 1 wherein the stem cell is altered at a plurality of genomic DNA sites by providing to the stem cell a plurality of guide RNA sequences complementary to different sites on genomic DNA of the eukaryotic cell,

providing to the eukaryotic cell a Cas 9 protein that interacts with the plurality of guide RNA sequences and cleaves the genomic DNA in a site specific manner.

8. The method of claim 1 further including the step of providing to the stem cell a donor sequence and inserting the donor sequence into the genomic DNA.

9. The method of claim 1 wherein the Cas 9 protein is human codon optimized.

10. The method of claim 1 wherein the Cas 9 protein includes a nuclear localization signal.

11. The method of claim 1 wherein the Cas 9 protein is a nickase.

12. The method of claim 1 wherein cleaving the genomic DNA in a site specific manner results in nonhomologous end joining.

13. The method of claim 1 wherein cleaving the genomic DNA in a site specific manner results in homologous recombination.