RNA-Guided Human Genome Engineering

• US 20140342458A1
• Filed: 06/30/2014
• Published: 11/20/2014
• 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
• Granted Patent: US 9,260,723 B2
• Time in Patent Office: Days
• US Class Current: 435/462
• 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 guide RNA sequence complementary to a target nucleic acid sequence and having a donor sequence hybridized thereto,

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 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 donor sequence is integrated into the genomic nucleic acid sequence.

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

3. The method of claim 1

wherein the guide RNA is provided to the cell by introducing to the cell a nucleic acid encoding the guide RNA,

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 including the transcriptional activator or repressor domain.

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 a mammalian cell.

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

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

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

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