# US 11,028,411 B2 · Systems and methods for one-shot guide RNA (ogRNA) targeting of endogenous and source DNA

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- Number: US 11,028,411 B2
- Document ID: US11028411B2
- Status: Granted
- Kind: B2
- Application number: 16/666,982
- Filed: 2019-10-29
- Published: 2020-02-20
- Granted: 2021-06-08
- Earliest claimed: 2016-12-05
- Assignees: Editas Medicine, Inc.
- Applicants: Editas Medicine, Inc.
- Inventors: Ari E. Friedland; Hariharan Jayaram; Barrett Ethan Steinberg
- Web page: https://patentgrep.com/patent/US11028411B2

## CPC

- C12N 15/907: {in mammalian cells}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 15/102: {Mutagenizing nucleic acids}
- 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 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- 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 2810/10: Vectors comprising a non-peptidic targeting moiety
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Engineered nucleic acids encoding genome editing system components are provided, as are engineered RNA-guided nucleases that include inserts encoded in part by cellular genomic or other sequences recognized by guide RNAs.

## Claims

### Claim 1

A method of altering a target site in a cell comprising delivering to the cell a transiently active genome editing system comprising a guide RNA (gRNA) and an engineered Cas9 protein, or a nucleic acid composition encoding the gRNA and the Cas9 protein, wherein
a) the gRNA comprises a targeting domain that is complementary to a eukaryotic nucleic acid sequence; and
b) the Cas9 protein is encoded by a nucleic acid comprising the eukaryotic nucleotide sequence and a protospacer adjacent motif (PAM), wherein the PAM is recognized by the Cas9 protein and is within or adjacent to the eukaryotic nucleotide sequence, and wherein the Cas9 protein comprises:
i) an amino acid insertion relative to SEQ ID NO: 2 selected from the group consisting of: E271_N272insGX6-10G, L371_N372insGX6-10G, and Q737_A738insGX6-10G; or
ii) an amino acid insertion S12_M13insGX6-10G relative to SEQ ID NO: 10,
wherein the amino acid insertion relative to SEQ ID NO:2 or SEQ ID NO: 10 results in a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 10, and having an RNA-guided nuclease activity.

### Claim 2 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein and the gRNA form a Cas9/gRNA complex.

### Claim 3 (depends on 2)

The method of claim 2, wherein the Cas9/gRNA complex is adapted to cleave the nucleic acid encoding the Cas9 protein.

### Claim 4 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein has at least about 80% nuclease activity of a wild-type Cas9 protein.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the engineered Cas9 protein comprises:
a) an insertion, relative to SEQ ID NO: 6, selected from the group consisting of:
c.813_814insN24-36,
c.1113_1114insN24-36, and
c.2211_2212insN24-36; or
b) an insertion c.36_37insN24-36 relative to SEQ ID NO: 11.

### Claim 6 (depends on 5)

The method of claim 5, wherein the nucleic acid encoding the engineered Cas9 protein comprises a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 11.

### Claim 7 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein is an engineered S. aureus Cas9.

### Claim 8 (depends on 1)

The method of claim 1, wherein the system alters both a cellular endogenous target gene and the RNA-guided nuclease expression.

### Claim 9 (depends on 1)

The method of claim 1, wherein the eukaryotic nucleic acid sequence is at least 17 nucleotides in length.

### Claim 10 (depends on 1)

The method of claim 1, wherein the targeting domain of the gRNA is 16-24 nucleotides in length.

### Claim 11 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is E271_N272insGX6-10G.

### Claim 12 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is L371_N372insGX6-10G.

### Claim 13 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 2, which is Q737_A738insGX6-10G.

### Claim 14 (depends on 1)

The method of claim 1, wherein the engineered Cas9 protein comprises an amino acid insertion relative to SEQ ID NO: 10, which is S12_M13insGX6-10G.

### Claim 15 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.813_814insN24-36.

### Claim 16 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.1113_1114insN24-36.

### Claim 17 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 6, which is c.2211_2212insN24-36.

### Claim 18 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the engineered Cas9 protein comprises an insertion relative to SEQ ID NO: 11, which is c.36_37insN24-36.

### Claim 19 (depends on 1)

The method of claim 1, wherein the nucleic acid composition comprises the nucleic acid encoding the Cas9 protein and a nucleic acid encoding the gRNA.

### Claim 20 (depends on 19)

The method of claim 19, wherein the nucleic acid encoding the Cas9 protein is present on a first vector, and the nucleic acid encoding the gRNA is present on a second vector.

### Claim 21 (depends on 20)

The method of claim 20, wherein one or both of the first and second vectors are viral vectors.

### Claim 22 (depends on 21)

The method of claim 21, wherein the viral vectors are selected from the group consisting of lentiviral vectors, adeno-associated virus (AAV) vectors, and combinations thereof.

### Claim 23 (depends on 21)

The method of claim 21, wherein the first and second viral vectors are adeno-associated virus (AAV) vectors.

### Claim 24 (depends on 1)

The method of claim 1, wherein the nucleic acid encoding the Cas9 protein further encodes the gRNA.

### Claim 25 (depends on 24)

The method of claim 24, wherein the nucleic acid is present on a vector.

### Claim 26 (depends on 25)

The method of claim 25, wherein the vector is a viral vector.

### Claim 27 (depends on 26)

The method of claim 26, wherein the viral vector is selected from the group consisting of lentiviral vectors, adeno-associated virus (AAV) vectors, and combinations thereof.

### Claim 28 (depends on 27)

The method of claim 27, wherein the viral vector is an adeno-associated virus (AAV) vector.