# US 11,111,492 B2 · Genome engineering methods using a cytosine-specific Cas9

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- Number: US 11,111,492 B2
- Document ID: US11111492B2
- Status: Granted
- Kind: B2
- Application number: 15/913,444
- Filed: 2018-03-06
- Published: 2018-09-20
- Granted: 2021-09-07
- Earliest claimed: 2017-03-06
- Assignees: The Florida State University Research Foundation, Inc.
- Applicants: Florida State University Research Foundation, Inc.
- Inventors: Hong Li; Martin Tsui; Travis Hand
- Web page: https://patentgrep.com/patent/US11111492B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- 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 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Described herein are methods and compositions for rapid and precise genome editing at any desired genomic position in almost all cells and organisms. The methods utilize a cytosine-specific Cas9 endonuclease and are suitable for applications requiring targeting cytosine adjacent DNA. The methods generally include introducing a cytosine-specific Cas9 endonuclease, a single guide RNA, and optionally a DNA repair template that is utilized in either non-homologous end joining or homology directed repair. The methods can be used to effect gene knockout, gene knockdown, gene substitution, or gene introduction in cells and organisms.

## Claims

### Claim 1

A method for specifically manipulating nucleic acids in a cell comprising:
contacting the cell with a cytosine-specific Cas9 endonuclease and a single guide RNA (sgRNA) targeting a protospacer sequence of the nucleic acids that is adjacent to a Protospacer Adjacent Motif (PAM) sequence of the nucleic acids,
wherein the cytosine-specific Cas9 endonuclease is a Type II-C Acidothermus cellulolyticus Cas9 (AceCas9).

### Claim 2 (depends on 1)

The method of claim 1, wherein the PAM sequence is downstream of the protospacer sequence on a non-targeting strand of the nucleic acids.

### Claim 3 (depends on 2)

The method of claim 2, wherein the PAM sequence is five nucleotides.

### Claim 4 (depends on 3)

The method of claim 3, wherein the PAM sequence is 5′-NNNCC-3′.

### Claim 5 (depends on 1)

The method of claim 1, wherein the sgRNA comprises an 18 to 26 nucleotide guide region complementary to the protospacer sequence.

### Claim 6 (depends on 1)

The method of claim 1, wherein an ambient temperature of the cell is 25-60° C.

### Claim 7 (depends on 1)

The method of claim 1, wherein an ambient temperature of the cell is 50-60° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the nucleic acids are deoxyribonucleic acids.

### Claim 9 (depends on 1)

The method of claim 1, wherein the nucleic acids are supercoiled or linearized.

### Claim 10 (depends on 1)

The method of claim 1, wherein a plasmid comprises the nucleic acids.

### Claim 11 (depends on 1)

The method of claim 1, wherein a chromosome comprises the nucleic acids.

### Claim 12 (depends on 1)

The method of claim 1, wherein the cell is a bacterial cell, a fungal cell, an archaea cell, a plant cell, or an animal cell.

### Claim 13 (depends on 1)

The method of claim 1, wherein contacting the cell with the cytosine-specific Cas9 endonuclease and the sgRNA introduces at least one nucleotide insertion and/or substitution in the nucleic acids.

### Claim 14 (depends on 13)

The method of claim 13, wherein contacting the cell with the cytosine-specific Cas9 endonuclease and the sgRNA causes disruption of a protein-encoding gene of the nucleic acids.

### Claim 15 (depends on 13)

The method of claim 13, further comprising contacting the cell with a desired nucleic acid repair template.

### Claim 16 (depends on 15)

The method of claim 15, wherein contacting the cell with the cytosine-specific Cas9 endonuclease, the sgRNA, and the desired nucleic acid repair template causes: (i) disruption of a protein-encoding gene of the nucleic acids, (ii) replacement of the protein-encoding gene of the nucleic acids with a substitute gene, or (iii) introduction a new gene into the nucleic acids.