# US 11,441,146 B2 · Compositions and methods for improving homogeneity of DNA generated using a CRISPR/Cas9 cleavage system

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- Number: US 11,441,146 B2
- Document ID: US11441146B2
- Status: Granted
- Kind: B2
- Application number: 15/402,833
- Filed: 2017-01-10
- Published: 2017-07-13
- Granted: 2022-09-13
- Earliest claimed: 2016-01-11
- Assignees: Christiana Care Health Services, Inc.
- Applicants: Christiana Care Health Services, Inc.
- Inventors: Eric Brian Kmiec; Pawel Alexander Bialk
- Web page: https://patentgrep.com/patent/US11441146B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to the unexpected discovery of a system and methods for precise homology directed repair after CRISPR/Cas9 cleavage. The invention includes a DNA cleavage and repair system comprising a CRISPR/Cas9 system and an oligonucleotide 100% complementary to cleaved DNA to promote homology directed DNA repair. The invention further includes methods for inducing homology directed repair of cleaved DNA and repairing a CRISPR/Cas9 cleavage.

## Claims

### Claim 1

A method of exchanging a single nucleotide in a DNA sequence using a CRISPR/Cas9 system in a synchronized and released population of cells in vitro, the method comprising:
cleaving a DNA within 20 bases of a single nucleotide target site in the synchronized and released population of cells using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex, thereby generating a cleaved DNA comprising a first cleavage end and a second cleavage end;
exchanging a single nucleotide at the single nucleotide target site by (a) annealing the cleaved DNA with a single-stranded deoxyoligonucleotide about 72 nucleotides in length comprising (i) a first region 100% complementary to the first cleavage end and at least one deoxynucleotide adjacent to the first cleavage end of the cleaved DNA, (ii) a second region 100% complementary to the second cleavage end and at least one deoxynucleotide adjacent to the second cleavage end of the cleaved DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the DNA and (b) fusing the first and second cleavage ends of the cleaved DNA with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first and second cleavage ends of the cleaved DNA to generate a repaired DNA at the single nucleotide target site comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first region of the single-stranded deoxyoligonucleotide is directly linked to the second region of the single-stranded deoxyoligonucleotide.

### Claim 3

A method of exchanging a single nucleotide in a DNA sequence cleaved by a CRISPR/Cas9 system in a synchronized and released population of cells in vitro, the method comprising:
exchanging a single nucleotide at a single nucleotide target site that is within 20 bases of the CRISPR/Cas9 cleavage site without introducing an insertion or deletion of one or more unwanted deoxynucleotides by (a) annealing a single-stranded oligonucleotide about 72 nucleotides in length to a DNA cleaved in one or more sites using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex in the synchronized and released population of cells and (b) fusing a first cleavage end and a second cleavage ends of the cleaved DNA with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first and second cleavage ends of the cleaved DNA to generate a repaired DNA comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide,
wherein the single-stranded oligonucleotide comprises (i) a first region 100% complementary to the first cleavage end and at least one deoxynucleotide adjacent to the first cleavage end of the cleaved DNA, (ii) a second region 100% complementary to the second cleavage end and at least one deoxynucleotide adjacent to the second cleavage end of the cleaved DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the DNA.

### Claim 4 (depends on 3)

The method of claim 3, wherein the one or more unwanted insertion or deletion is selected from the group consisting of: a single deoxynucleotide, and two or more deoxynucleotides.

### Claim 5 (depends on 3)

The method of claim 3, wherein the at least one deoxynucleotide adjacent to the first cleavage end and the at least one deoxynucleotide adjacent to the second generated cleavage end independently comprise about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 deoxynucleotides.

### Claim 6 (depends on 3)

The method of claim 3, wherein the first region of the single-stranded oligonucleotide is directly linked to the second region of the single-stranded oligonucleotide.

### Claim 7 (depends on 1)

The method of claim 1, wherein no collateral on-site or off-site DNA mutagenesis is produced.

### Claim 8 (depends on 3)

The method of claim 3, wherein no collateral on-site or off-site DNA mutagenesis is produced.