Genome engineering

Patent metadata

Field	Value
Publication number	US-10563225-B2
Application number	US-201414319380-A
Country	US
Kind code	B2
Filing date	Jun 30, 2014
Priority date	Jul 26, 2013
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

Methods are provided for the use of Cas9 in genome engineering of stem cells. Methods include introducing into the stem cell a first foreign nucleic acid encoding a guide RNA complementary to a target DNA and which guides a Cas9 enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, introducing into the stem cell a second foreign nucleic acid encoding a donor nucleic acid sequence, wherein the guide RNA and the donor nucleic acid sequences are expressed, wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the stem cell.

First claim

The invention claimed is:

1. A method of altering target DNA in a human induced pluripotent stem cell in vitro comprising providing to the stem cell a Cas 9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner,
introducing into the stem cell a guide RNA complementary to the target DNA and which guides the enzyme to the target DNA, wherein the guide RNA and the enzyme are members of a co-localization complex for the target DNA,
introducing into the stem cell a donor nucleic acid sequence, wherein the guide RNA and the donor nucleic acid sequence are linked,
wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid sequence is inserted into the target DNA to produce altered DNA in the stem cell.

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

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

4. The method of claim 1 wherein the guide RNA is a crRNA-tracrRNA fusion.

5. The method of claim 1 wherein the donor nucleic acid sequence is inserted by homologous recombination.

6. The method of claim 1 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.

7. The method of claim 1 wherein the guide RNA is introduced into the stem cell as a first foreign nucleic acid which is expressed by the stem cell.

8. The method of claim 1 further comprising introducing multiple donor nucleic acid sequences and multiple guide RNAs to produce multiple alterations to the DNA in the cell.

9. The method of claim 1 wherein the Cas9 enzyme is S. pyogenes Cas9.

10. The method of claim 1 wherein the target DNA is genomic DNA.

11. The method of claim 1 wherein the guide RNA is about 100 nucleotides.

Assignees

• Harvard College

Inventors

• Church George M
• Yang Luhan
• Guell Marc
• Yang Joyce Lichi

Classifications

• C12N15/85 Primary

  for animal cells · CPC title

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N2830/003

  tet inducible · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title