# US 11,028,380 B2 · CAS9-CAS9 fusion proteins

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- Number: US 11,028,380 B2
- Document ID: US11028380B2
- Status: Granted
- Kind: B2
- Application number: 16/226,202
- Filed: 2018-12-19
- Published: 2019-09-12
- Granted: 2021-06-08
- Earliest claimed: 2014-12-22
- Assignees: University of Massachusetts
- Applicants: University of Massachusetts
- Inventors: Scot Andrew Wolfe; Mehmet Fatih Bolukbasi; Ankit Gupta; Erik J Sontheimer; Nadia Amrani
- Web page: https://patentgrep.com/patent/US11028380B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- 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)
- 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)
- C12Y: ENZYMES

## Abstract

The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

## Claims

### Claim 1

A fusion protein comprising a first Cas9 nuclease, said first nuclease comprising a protospacer adjacent motif recognition domain having a lysine-substituted, alanine-substituted or serine-substituted arginine residue and a second Cas9 nuclease.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein said first and second Cas9 nucleases are selected from the group consisting of Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9) and Actinomyces naeslundii Cas9 (AnCas9).

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein said substituted arginine protospacer adjacent motif (PAM) recognition residue participates in a base-specific binding or contacts a phosphodiester backbone.

### Claim 4 (depends on 3)

The fusion protein of claim 3, wherein said substituted arginine PAM recognition residue participates in a base-specific binding.

### Claim 5 (depends on 3)

The fusion protein of claim 3, wherein said substituted arginine PAM recognition residue contacts a phosphodiester backbone residue.

### Claim 6 (depends on 4)

The fusion protein of claim 4, wherein the first Cas9 nuclease has a single point mutation.

### Claim 7 (depends on 5)

The fusion protein of claim 5, wherein the first Cas9 nuclease has a double mutation.

### Claim 8 (depends on 1)

The fusion protein of claim 1, wherein said first Cas9 nuclease is selected from the group consisting of SpCas9R1333K, SpCas9R1333S, SpCas9R1335K, NmCas9R1025A and NmCas9K1013A/R1025A.

### Claim 9 (depends on 1)

The fusion protein of claim 1, wherein said second Cas9 nuclease is selected from the group consisting of a Cas9 nickase and a nuclease-dead Cas9 (dCas9).

### Claim 10 (depends on 1)

The fusion protein of claim 1, wherein said second Cas9 nuclease is selected from the group consisting of nuclease-dead NmCas9 (NmdCas9), NmCas9 nuclease, NmCas9 HNH nickase, and NmCas9 RuvC nickase.

### Claim 11 (depends on 1)

The fusion protein of claim 1, wherein said first and second Cas9 nucleases are bound to a guide RNA comprising a guide sequence element.

### Claim 12 (depends on 11)

The fusion protein of claim 11, wherein said guide sequence element is truncated.

### Claim 13 (depends on 12)

The fusion protein of claim 12, wherein said truncated guide sequence element is less than twenty nucleotides.