# US 11,306,328 B2 · Genome engineering

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,306,328 B2
- Document ID: US11306328B2
- Status: Granted
- Kind: B2
- Application number: 14/319,498
- Filed: 2014-06-30
- Published: 2015-01-29
- Granted: 2022-04-19
- Earliest claimed: 2013-07-26
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: George M. Church; Luhan Yang; Marc Guell; Joyce Lichi Yang
- Web page: https://patentgrep.com/patent/US11306328B2

## CPC

- C12N 15/907: {in mammalian cells}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- C07H 21/02: with ribosyl as saccharide radical
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 2510/00: Genetically modified cells
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2800/90: Vectors containing a transposable element
- C12N 2830/003: tet inducible
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

The method of claim 1 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNAs to produce multiple alterations to the DNA in the cell.

### Claim 8 (depends on 1)

The method of claim 1 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

### Claim 9 (depends on 1)

The method of claim 1 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

The method of claim 1 wherein the eukaryotic cell is a human induced pluripotent stem cell.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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

### Claim 14

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 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 modified from native form to include a 5′ Cap structure,
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.

### Claim 15 (depends on 14)

The method of claim 14 wherein the guide RNA is between about 10 and about 500 nucleotides.

### Claim 16 (depends on 14)

The method of claim 14 wherein the guide RNA is between about 20 and about 100 nucleotides.

### Claim 17 (depends on 14)

The method of claim 14 wherein the RNA is a crRNA-tracrRNA fusion.

### Claim 18 (depends on 14)

The method of claim 14 wherein the donor nucleic acid sequence is inserted by homologous recombination.

### Claim 19 (depends on 14)

The method of claim 14 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.

### Claim 20 (depends on 14)

The method of claim 14 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNA to produce multiple alterations to the DNA in the cell.

### Claim 21 (depends on 14)

The method of claim 14 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

### Claim 22 (depends on 14)

The method of claim 14 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

### Claim 23 (depends on 14)

The method of claim 14 wherein the eukaryotic cell is a human induced pluripotent stem cell.

### Claim 24 (depends on 14)

The method of claim 14 wherein the guide RNA is about 100 nucleotides.

### Claim 25 (depends on 14)

The method of claim 14 wherein the eukaryotic cell is a stem cell.

### Claim 26

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 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 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.

### Claim 27 (depends on 26)

The method of claim 26 wherein the guide RNA is between about 10 and about 500 nucleotides.

### Claim 28 (depends on 26)

The method of claim 26 wherein the guide RNA is between about 20 and about 100 nucleotides.

### Claim 29 (depends on 26)

The method of claim 26 wherein the RNA is a crRNA-tracrRNA fusion.

### Claim 30 (depends on 26)

The method of claim 26 further comprising in steps (a) and (b) introducing multiple donor nucleic acids and multiple guide RNA to produce multiple alterations to the DNA in the cell.

### Claim 31 (depends on 26)

The method of claim 26 wherein after producing altered DNA in the cell, the nucleic acid encoding the Cas 9 enzyme that forms a co-localization complex with the guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is removed from the cell genome.

### Claim 32 (depends on 26)

The method of claim 26 wherein the guide RNA is physically attached to the donor nucleic acid sequence.

### Claim 33 (depends on 26)

The method of claim 26 wherein the eukaryotic cell is a human induced pluripotent stem cell.

### Claim 34 (depends on 26)

The method of claim 26 wherein the guide RNA is about 100 nucleotides.

### Claim 35 (depends on 26)

The method of claim 26 wherein the eukaryotic cell is a stem cell.