Multiplexed genome editing

Patent metadata

Field	Value
Publication number	US-10375938-B2
Application number	US-201815888581-A
Country	US
Kind code	B2
Filing date	Feb 5, 2018
Priority date	Oct 8, 2015
Publication date	Aug 13, 2019
Grant date	Aug 13, 2019

Abstract

A method of modulating some or all copies of a gene in a cell is provided including introducing into a cell one or more ribonucleic acid (RNA) sequences that comprise a portion that is complementary to all or a portion of each of the one or more target nucleic acid sequences, and a nucleic acid sequence that encodes a Cas protein and maintaining the cells under conditions in which the Cas protein is expressed and the Cas protein binds and modulates the one or more target nucleic acid sequences in the cell.

First claim

The invention claimed is:

1. A method of altering porcine endogenous retrovirus (PERV) pol genes in a cell comprising:
introducing into the cell a Cas protein and guide RNAs, wherein each guide RNA comprises a sequence that is complementary to a portion of a PERV pol gene; and
maintaining the cell under conditions in which the guide RNAs and the Cas protein form co-localization complexes with a plurality of PERV pol genes and the Cas protein cuts at least 20% of the PERV pol genes.

2. The method of claim 1 , wherein the cuts to the PERV pol genes result in
indels or mutations.

3. The method of claim 1 , wherein the cell is a mammalian cell.

4. The method of claim 1 , wherein the cell is from an embryo.

5. The method of claim 1 , wherein the cell is a stem cell, zygote, or a germ line cell.

6. The method of claim 1 , wherein the cell is an embryonic stem cell or pluripotent stem cell.

7. The method of claim 1 , wherein the cell is a somatic cell.

8. The method of claim 1 , wherein the cell is a eukaryotic cell.

9. The method of claim 1 , wherein the cell is an animal cell.

10. The method of claim 1 , wherein the cell is a porcine cell.

11. The method of claim 1 , wherein all copies of the PERV pol genes are cut.

12. The method of claim 1 , wherein at least 97% of the PERV pol genes are cut.

13. The method of claim 12 , wherein the cuts to the pol genes result in indels or mutations.

14. The method of claim 13 , wherein the cell has at least a 1000-fold reduction in PERV transmission to a second cell compared to a wild type control cell.

15. The method of claim 14 , wherein the second cell is a human cell.

16. The method of claim 1 , wherein the cuts present in at least 20% of PERV pol genes are determined by sequencing.

17. The method of claim 1 , wherein the cell contains intact PERV gag and/or env genes.

18. The method of claim 1 , wherein at least 50% of the PERV pol genes comprise genomic indels.

19. The method of claim 1 , wherein at least 80% of the PERV pol genes comprise genomic indels.

Assignees

• Harvard College

Inventors

• Church George M
• Yang Luhan
• Guell Marc

Classifications

• C12N15/8509 Primary

  for producing genetically modified animals, e.g. transgenic · CPC title

• A01K2267/025

  Animal producing cells or organs for transplantation · CPC title

• C12N15/85

  for animal cells · CPC title

• A01K67/0276

  Knock-out vertebrates · CPC title

• A01K2207/10

  Animals modified by protein administration, for non-therapeutic purpose · CPC title