RNA-guided human genome engineering

Patent metadata

Field	Value
Publication number	US-9260723-B2
Application number	US-201414319255-A
Country	US
Kind code	B2
Filing date	Jun 30, 2014
Priority date	Dec 17, 2012
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

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.

First claim

The invention claimed is:

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.

Assignees

• Harvard College

Inventors

• Mali Prashant G
• Church George M
• Yang Luhan

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N9/22 Primary

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

• C12N2310/20

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

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title