# US 10,767,194 B2 · RNA-guided transcriptional regulation

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- Number: US 10,767,194 B2
- Document ID: US10767194B2
- Status: Granted
- Kind: B2
- Application number: 16/441,209
- Filed: 2019-06-14
- Published: 2020-01-23
- Granted: 2020-09-08
- Earliest claimed: 2013-06-04
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: George M. Church; Prashant G. Mali; Kevin M. Esvelt
- Web page: https://patentgrep.com/patent/US10767194B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/907: {in mammalian cells}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3513: Protein; Peptide
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

## Claims

### Claim 1

A method of inserting a donor nucleic acid sequence into a cell using homologous recombination comprising
providing to the cell two guide RNAs with each guide RNA having a spacer sequence, a tracr mate sequence and a tracr sequence, and with a portion of the tracr sequence being hybridized to the tracr mate sequence and with the tracr mate sequence and the tracr sequence being linked by a linker nucleic acid sequence and with each spacer sequence being complementary to an adjacent site in a DNA target nucleic acid,
providing to the cell a donor nucleic acid sequence,
providing to the cell a Cas9 protein nickase, and
wherein each of the two guide RNAs co-localize with the Cas9 protein nickase to the DNA target nucleic acid resulting in an offset nick with 5′-overhangs, and
wherein the donor nucleic acid sequence is inserted into the target nucleic acid at the offset nick using homologous recombination.

### Claim 2 (depends on 1)

The method of claim 1
wherein the two guide RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid encoding the two guide RNAs,
wherein the Cas9 protein nickase is provided to the cell by introducing into the cell a second foreign nucleic acid encoding the Cas9 protein, and
wherein the two guide RNAs and the Cas9 protein nickase are expressed.

### Claim 3 (depends on 1)

The method of claim 1 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.

### Claim 4 (depends on 1)

The method of claim 1 wherein the tracr sequence is between 90 to 500 nucleotides.

### Claim 5 (depends on 1)

The method of claim 1 wherein the tracr sequence is between 90 to 200 nucleotides.

### Claim 6 (depends on 1)

The method of claim 1 wherein the tracr sequence is between 100 to 200 nucleotides.