RNA-guided human genome engineering

• US 9,260,723 B2
• Filed: 06/30/2014
• Issued: 02/16/2016
• Est. Priority Date: 12/17/2012
• Status: Active Grant

• 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
• Primary Examiner(s): Yu, Misook
• Assistant Examiner(s): WU, JULIE ZHEN QIN
• Application Number:
  US14/319,255
• Publication Number:
  US 20140342458A1
• Time in Patent Office: 596 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 integrating foreign DNA into a genomic nucleic acid sequence of a eukaryotic cell comprising providing to the eukaryotic cell a single stranded DNA (ssDNA) donor sequence tethered to a guide RNA sequence via hybridization, wherein the guide RNA sequence comprises a spacer sequence complementary to a target nucleic acid sequence, providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the spacer sequence of the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner;

and wherein the ssDNA donor sequence is integrated into the genomic nucleic acid sequence.

2. The method of claim 1 wherein the ssDNA donor nucleic acid is integrated into the genomic nucleic acid sequence by homologous recombination.

3. The method of claim 1 wherein the guide RNA sequence is provided to the cell by introducing to the cell a nucleic acid encoding the guide RNA sequence, wherein the Cas9 enzyme is provided to the cell by introducing to the cell a nucleic acid encoding the Cas9 enzyme, and wherein the cell expresses the guide RNA and the Cas9 protein.

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

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

6. The method or claim 1 wherein the eukaryotic cell is a human cell.

7. The method of claim 1 wherein the guide RNA sequence is between about 10 to about 250 nucleotides.

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

9. The method of claim 1 wherein the guide RNA sequence is between about 100 to about 250 nucleotides.