# US 11,306,327 B1 · Nucleic acid-guided nucleases

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- Number: US 11,306,327 B1
- Document ID: US11306327B1
- Status: Granted
- Kind: B1
- Application number: 17/554,736
- Filed: 2021-12-17
- Granted: 2022-04-19
- Earliest claimed: 2017-06-23
- Assignees: Inscripta, Inc.
- Applicants: Inscripta, Inc.
- Inventors: Andrew Garst; Ryan T. Gill; Tanya Elizabeth Warnecke Lipscomb
- Web page: https://patentgrep.com/patent/US11306327B1

## CPC

- C12N 15/902: {using homologous recombination}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- 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}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/81: for yeasts
- C12N 15/85: for animal cells
- C12N 15/905: {in yeast}
- C12N 15/907: {in mammalian cells}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed herein are nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Disclosed herein are engineered non-naturally occurring nucleic acid-guided nucleases, guide nucleic acids, and targetable nuclease systems, and methods of use. Targetable nuclease systems can be used to edit genetic targets, including recursive genetic engineering and trackable genetic engineering methods.

## Claims

### Claim 1

A nucleic acid-guided nuclease system comprising:
(a) a nucleic acid having at least 90% identity to SEQ ID NO: 24 or SEQ ID NO: 44, wherein the nucleic acid encodes a nucleic acid-guided nuclease comprising the amino acid sequence of SEQ ID No. 4;
(b) an engineered guide nucleic acid sequence capable of complexing with the nucleic acid-guided nuclease, wherein the engineered guide nucleic acid sequence is configured to hybridize to a TTTN sequence of a target region in a genome of a cell;
(c) an editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell;
wherein the system results in a genome edit in the target region in the genome of the cell facilitated by the nuclease, the engineered guide nucleic acid, and the editing sequence.

### Claim 2 (depends on 1)

The system of claim 1, wherein the engineered guide nucleic acid and the editing sequence are provided as a single nucleic acid.

### Claim 3 (depends on 1)

The system of claim 1, wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for E. coli.

### Claim 4 (depends on 1)

The system of claim 1, wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for S. cerevisiae.

### Claim 5 (depends on 1)

The system of claim 1, wherein the nucleic acid encoding the nucleic acid-guided nuclease is codon optimized for mammalian cells.

### Claim 6 (depends on 1)

The system of claim 1, wherein the target region is within a coding region of a protein.

### Claim 7 (depends on 1)

The system of claim 1, wherein the target region is within a non-coding region of a protein.

### Claim 8 (depends on 7)

The system of claim 7, wherein the target region is within a regulatory region of a protein.

### Claim 9 (depends on 1)

The system of claim 1, wherein the editing sequence having a change in sequence relative to the sequence of a target region in a genome of a cell further comprises a mutation in a protospacer adjacent motif (PAM) sequence.

### Claim 10 (depends on 1)

A method of modifying a target region in the genome of a cell, the method comprising:
(a) contacting a cell with: the nucleic acid-guided nuclease system of claim 1; and
(b) allowing the nucleic-acid guided nuclease system to create a genome edit in a target region of the genome of the cell.

### Claim 11 (depends on 10)

The method of claim 10, wherein the engineered guide nucleic acid sequence and the editing sequence are provided as a single nucleic acid.

### Claim 12 (depends on 10)

The method of claim 10, wherein the nucleic acid having at least 90% identity to SEQ ID No. 44 or SEQ ID No. 24 is codon optimized for the cell to be edited.

### Claim 13 (depends on 12)

The method of claim 12, wherein the nucleic acid-guided nuclease is codon optimized for E. coli.

### Claim 14 (depends on 12)

The method of claim 12, wherein the nucleic acid-guided nuclease is codon optimized for S. cerevisiae.

### Claim 15 (depends on 12)

The method of claim 12, wherein the nucleic acid-guided nuclease is codon optimized for mammalian cells.

### Claim 16 (depends on 10)

The method of claim 10, wherein the editing sequence encoding a nucleic acid complementary to the target region having a change in sequence relative to the target region further comprises a mutation in a protospacer adjacent motif (PAM) site.

### Claim 17 (depends on 10)

The method of claim 10, wherein the target region is within a eukaryotic cell.

### Claim 18 (depends on 10)

The method of claim 10, wherein the target region is within a bacterial cell.

### Claim 19 (depends on 10)

The method of claim 10, wherein the target region is within a plant cell.

### Claim 20 (depends on 10)

The method of claim 10, wherein the target region is within a mammalian cell.