Genome engineering

Patent metadata

Field	Value
Publication number	US-12473575-B2
Application number	US-202217715177-A
Country	US
Kind code	B2
Filing date	Apr 7, 2022
Priority date	Jul 26, 2013
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

Methods are provided for altering target DNA in a cell genetically modified to express 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. Methods include introducing into the cell a first foreign nucleic acid encoding a donor nucleic acid sequence, introducing into the cell from media surrounding the cell the guide RNA complementary to the target DNA and which guides the Cas 9 enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, wherein the donor nucleic acid sequence is 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 cell.

First claim

The invention claimed is:

1 . A method of multiplex altering of a plurality of target DNA at multiple genomic loci in a eukaryotic cell in vitro wherein the cell genome is genetically modified to include a nucleic acid encoding a Cas 9 enzyme and wherein the Cas 9 enzyme is expressed, comprising
(a) introducing into the cell a DNA donor nucleic acid sequence, (b) introducing into the cell a guide RNA complementary to the target DNA, wherein the guide RNA and the Cas 9 enzyme are members of a co-localization complex for the target DNA, wherein the guide RNA is in native form or is modified from native form to include a 5′ Cap structure or is modified from native form by treating with phosphatase, 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 cell, and repeating steps (a) and (b) to result in multiple exogenous nucleic acid insertions in the cell wherein steps (a) and (b) are carried out without further introducing a nucleic acid encoding a Cas 9 enzyme.

2 . A eukaryotic cell including a first foreign nucleic acid integrated into the genome of the cell and encoding for a Cas protein of a Type II CRISPR system, wherein the first foreign nucleic acid encoding for the Cas protein is expressed and forms a co-localization complex with guide RNA complementary to target DNA.

3 . The eukaryotic cell of claim 2 wherein the Cas protein is a Cas9 enzyme, a Cas9 nickase or a nuclease null Cas9.

4 . The eukaryotic cell of claim 2 further including a second foreign nucleic acid encoding for a guide RNA complementary to the target DNA and which guides the Cas protein to the target DNA, wherein the guide RNA and the Cas protein are members of a co-localization complex for the target DNA.

5 . The eukaryotic cell of claim 2 further including a donor nucleic acid sequence.

6 . The eukaryotic cell of claim 5 wherein at least one donor nucleic acid sequence is inserted into the genome of the cell.

7 . The eukaryotic cell of claim 6 wherein the at least one donor nucleic acid sequence is a single stranded donor nucleic acid sequence.

8 . The eukaryotic cell of claim 2 further including an inducible promoter for promoting expression of the Cas protein.

9 . The eukaryotic cell of claim 2 wherein the cell has been genetically modified by inserting an enzyme removable vector or cassette including the integrated first foreign nucleic acid encoding the Cas protein into genomic DNA of the cell.

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

11 . The eukaryotic cell of claim 2 wherein the guide RNA is between about 20 to about 100 nucleotides.

12 . The eukaryotic cell of claim 2 wherein the guide RNA is between about 100 to about 500 nucleotides.

13 . The eukaryotic cell of claim 2 wherein the guide RNA is a tracrRNA-crRNA fusion.

14 . The eukaryotic cell of claim 2 wherein the target DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

15 . The eukaryotic cell of claim 2 wherein the first foreign nucleic acid is reversibly inserted into genomic DNA of the eukaryotic cell.

16 . The eukaryotic cell of claim 2 further including multiple guide RNAs and multiple donor nucleic acid sequences.

17 . The eukaryotic cell of claim 16 wherein multiple donor nucleic acid sequences are inserted into the genome of the cell.

18 . The eukaryotic cell of claim 17 wherein the multiple donor nucleic acid sequences are single stranded donor nucleic acid sequences.

19 . The eukaryotic cell of claim 2 wherein the first foreign nucleic acid is removable from genomic DNA of the eukaryotic cell using a transposase.

20 . The eukaryotic cell of claim 2 wherein the eukaryotic cell is a yeast cell, a plant cell or an animal cell.

21 . The eukaryotic cell of claim 2 wherein the eukaryotic cell is a human cell.

22 . The eukaryotic cell of claim 2 wherein the eukaryotic cell is a stem cell.

23 . The eukaryotic cell of claim 2 wherein the eukaryotic cell is a human stem cell.

24 . The eukaryotic cell of claim 2 wherein the eukaryotic cell is a human induced pluripotent stem cell.

25 . The eukaryotic cell of claim 2 wherein the guide RNA and the donor nucleic acid sequence are linked, or wherein the guide RNA and the donor nucleic acid sequence are physically attached.

26 . The eukaryotic cell of claim 2 wherein the Cas protein is an S. pyogenes Cas protein.

Assignees

• Harvard College

Inventors

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

Classifications

• C12N15/86

  Viral vectors · CPC title

• C12N2510/00

  Genetically modified cells · CPC title

• C12N5/0696

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

• C12N2830/003

  tet inducible · CPC title

• C12N2800/90

  Vectors containing a transposable element · CPC title