# US 11,299,732 B2 · Compositions and methods for transcription-based CRISPR-Cas DNA editing

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

- Number: US 11,299,732 B2
- Document ID: US11299732B2
- Status: Granted
- Kind: B2
- Application number: 15/501,640
- Filed: 2015-08-07
- Published: 2017-08-17
- Granted: 2022-04-12
- Earliest claimed: 2014-08-07
- Assignees: The Rockefeller University
- Applicants: The Rockefeller University
- Inventors: Luciano Marraffini; Gregory Goldberg; Poulami Samai
- Web page: https://patentgrep.com/patent/US11299732B2

## CPC

- 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 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are compositions and methods for cleaving a DNA sequence in a cell. The methods involve comprising introducing into a cell a recombinant vector containing a clustered regularly interspaced short palindromic repeats (CRISPR) system. The system includes a CRISPR RNA (crRNA) targeted to a DNA sequence in the cell that is operatively linked to a promoter; and CRISPR-associated enzymes (Cas) 10, Cas6, and at least one Csm protein. The Cas 10 cleaves the DNA sequence only during transcription of the DNA sequence that is operatively linked to the promoter. Also provided are recombinant vectors for modifying cells, cells that contain the recombinant vectors and modifications introduced by them, and kits that include the modified vectors.

## Claims

### Claim 1

A method for transcription-restricted DNA modification that modifies a DNA sequence in a chromosome of a cell, the method comprising modifying the DNA sequence in the chromosome of the cell only during transcription of said DNA sequence, and wherein the modifying comprises cutting only a non-template strand only within said DNA sequence, by expressing in the cell from a recombinant vector a clustered regularly interspaced short palindromic repeats (CRISPR) system, the CRISPR system comprising nucleotide sequences encoding: i) a CRISPR RNA (crRNA) targeted only to a DNA sequence on a coding strand of the DNA sequence that is transcribed and is operatively linked to a promoter; and CRISPR-associated enzymes (Cas) 10, Cas6, Csm2, Csm3, Csm4, Csm5 and Csm6, the method further comprising determining the DNA sequence that was transcribed and modified.

### Claim 2 (depends on 1)

The method of claim 1, wherein the modification is a conditional modification of the DNA such that transcription of the DNA from the promoter is not constitutive transcription.

### Claim 3 (depends on 1)

The method of claim 1, wherein the promoter is an inducible promoter.

### Claim 4 (depends on 3)

The method of claim 3, further comprising inducing transcription from the inducible promoter such that the DNA modification takes place only during the induced transcription.

### Claim 5 (depends on 1)

The method of claim 1, wherein the modification of the DNA sequence confers a change in phenotype of the cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the modification of the DNA sequence is lethal to the cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the DNA sequence encodes a selectable marker, or a detectable marker.