# US 11,111,508 B2 · Modified CAS9 compositions and methods of use

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- Number: US 11,111,508 B2
- Document ID: US11111508B2
- Status: Granted
- Kind: B2
- Application number: 15/762,025
- Filed: 2016-10-25
- Published: 2018-09-20
- Granted: 2021-09-07
- Earliest claimed: 2015-10-30
- Assignees: Brandeis University
- Applicants: Brandeis University
- Inventors: Nelson Lau
- Web page: https://patentgrep.com/patent/US11111508B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/907: {in mammalian cells}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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}
- C07K 2319/00: Fusion polypeptide
- 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)
- 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

The invention features Cas9 fusion polypeptides. In one embodiment of the invention, Cas9 is fused to a SNAP tag that enhances Cas9's gene repair function.

## Claims

### Claim 1

A Cas9 fusion polypeptide having the sequence of SEQ ID NO:2.

### Claim 2 (depends on 1)

A polynucleotide encoding the polypeptide of claim 1.

### Claim 3 (depends on 2)

An expression vector comprising the polynucleotide of claim 2.

### Claim 4 (depends on 3)

A cell comprising the expression vector of claim 3.

### Claim 5 (depends on 1)

A method for enhancing transgene integration efficiency, the method comprising expressing in a cell a vector encoding the polypeptide of claim 1, a small guide RNA, and a transgene suitable for integration into a genome.

### Claim 6 (depends on 5)

The method of claim 5, wherein the integration efficiency is increased at least about 2-fold relative to the level present in a corresponding control cell expressing wild-type Cas9.

### Claim 7 (depends on 1)

A method for enhancing Homologous DNA Recombination (HDR), the method comprising expressing in a cell vector encoding the polypeptide of claim 1, a small guide RNA, and a transgene suitable for integration into a genome.