# US 10,767,193 B2 · Engineered CAS9 systems for eukaryotic genome modification

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,767,193 B2
- Document ID: US10767193B2
- Status: Granted
- Kind: B2
- Application number: 16/277,823
- Filed: 2019-02-15
- Published: 2019-08-15
- Granted: 2020-09-08
- Earliest claimed: 2018-02-15
- Assignees: Sigma-Aldrich Co.
- Applicants: Sigma-Aldrich Co., LLC
- Inventors: Timothy Seebeck; Fuqiang Chen; Gregory D. Davis
- Web page: https://patentgrep.com/patent/US10767193B2

## 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 15/102: {Mutagenizing nucleic acids}
- 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 15/85: for animal cells
- C07K 2319/00: Fusion polypeptide
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12Y 301/21001: Deoxyribonuclease I (3.1.21.1)
- 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

Engineered Cas9 systems that utilize alternate protospacer adjacent motifs for target DNA binding, nucleic acids encoding the engineered Cas9 systems, and methods of using the engineered Cas9 systems for modifying target chromosomal sequences in eukaryotic cells.

## Claims

### Claim 1

A system for eukaryotic genome modification comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS), and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T.

### Claim 2 (depends on 1)

The system of claim 1, wherein the engineered Bacillus smithii Cas9 protein further comprises at least one heterologous domain and the at least one heterologous domain is a cell-penetrating domain, a marker domain, a chromatin modulating motif, an RNA aptamer binding domain, or combination thereof.

### Claim 3 (depends on 1)

The system of claim 1, wherein the engineered Bacillus smithii Cas9 protein further comprises (i) at least one modification within a RuvC domain or an HNH domain, such that the engineered Bacillus smithii Cas9 protein functions as a nickase; or (ii) at least one modification within a RuvC domain and an HNH domain, such that the engineered Bacillus smithii Cas9 protein is catalytically inactive.

### Claim 4 (depends on 1)

The system of claim 1, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:31.

### Claim 5 (depends on 1)

The system of claim 1, wherein the engineered Bacillus smithii Cas9 protein further comprises a chromatin modulating motif.

### Claim 6 (depends on 1)

The system of claim 1, wherein the engineered Bacillus smithii Cas9 protein comprises at least one modification within a RuvC domain or an HNH domain, such that the engineered Bacillus smithii Cas9 protein functions as a nickase, and wherein the engineered Bacillus smithii Cas9 protein further comprises a cell-penetrating domain, a marker domain, an RNA aptamer binding domain, or combination thereof.

### Claim 7 (depends on 1)

The system of claim 1, wherein the engineered Bacillus smithii Cas9 protein comprises at least one modification within a RuvC domain and an HNH domain, such that the engineered Bacillus smithii Cas9 protein is catalytically inactive, and wherein the engineered Bacillus smithii Cas9 protein further comprises a cell-penetrating domain, a marker domain, a chromatin modulating motif, an epigenetic modification domain, a transcriptional regulation domain, an RNA aptamer binding domain, or combination thereof.

### Claim 8 (depends on 1)

The system of claim 1, wherein the system is selected from the group consisting of:
(1) a system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a NLS, which comprises the amino acid sequence of SEQ ID NO: 2; and (b) an engineered guide RNA, which comprises the nucleotide sequence of SEQ ID NO: 31 designed to hybridize to a target sequence 5′ to a PAM comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T; and
(2) a system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a NLS, which comprises the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120; and (b) an engineered guide RNA designed to hybridize to a target sequence 5′ to a PAM comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T.

### Claim 9

A plurality of nucleic acids encoding a system for eukaryotic genome modification comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T; the plurality of nucleic acids comprising at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein, and at least one nucleic acid encoding the engineered guide RNA.

### Claim 10 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein is RNA.

### Claim 11 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein is DNA.

### Claim 12 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein is codon optimized for expression in a eukaryotic cell.

### Claim 13 (depends on 12)

The plurality of nucleic acids of claim 12, wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

### Claim 14 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered guide RNA is DNA.

### Claim 15 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein is operably linked to a phage promoter sequence for in vitro RNA synthesis or protein expression in a bacterial cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a phage promoter sequence for in vitro RNA synthesis.

### Claim 16 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the at least one nucleic acid encoding the engineered Bacillus smithii Cas9 protein is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell.

### Claim 17 (depends on 9)

The plurality of nucleic acids of claim 9, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:31.

### Claim 18 (depends on 9)

At least one vector comprising the plurality of nucleic acids of claim 9, wherein the at least one vector is a plasmid vector, a viral vector, or a self-replicating viral RNA replicon.

### Claim 19

A eukaryotic cell comprising at least one system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T, wherein the eukaryotic cell is a non-human mammalian cell, a plant cell, a non-mammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism and wherein the eukaryotic cell is in vivo, ex vivo, or in vitro.

### Claim 20

A human eukaryotic cell comprising at least one system comprising (a) an engineered Bacillus smithii Cas9 protein comprising a nuclear localization signal (NLS); and (b) an engineered guide RNA, wherein the engineered guide RNA is designed to complex with the engineered Bacillus smithii Cas9 protein and the engineered guide RNA comprises a 5′ guide sequence designed to hybridize with a target sequence in a double-stranded sequence, and wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein the human eukaryotic cell is ex vivo, or in vitro.

### Claim 21

A fusion protein comprising an engineered Bacillus smithii Cas9 protein linked to at least one chromatin modulating motif,
wherein the chromatin modulating motif is a high mobility group (HMG) box (HMGB) DNA binding domain, a HMG nucleosome-binding (HMGN) protein, a central globular domain from a histone H1 variant comprising SEQ ID NO: 74, or a combination thereof,
wherein the at least one chromatin modulating motif is linked to the Cas9 protein at its N-terminus, C-terminus, or a combination thereof,
wherein, together with an engineered guide RNA designed to complex with the Cas9 protein, the fusion protein is capable of hybridizing with a target sequence in a double-stranded sequence, and
wherein the target sequence is 5′ to a protospacer adjacent motif (PAM) comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T.

### Claim 22 (depends on 21)

The fusion protein of claim 21, wherein the at least one chromatin modulating motif is a human HMGB1 box A domain, a HMGN1 protein, a human histone H1 central globular domain comprising SEQ ID NO: 74, or a combination thereof.

### Claim 23 (depends on 21)

The fusion protein of claim 21, wherein the at least one chromatin modulating motif is linked to the Cas9 protein directly via a chemical bond, indirectly via a linker, or a combination thereof.

### Claim 24 (depends on 21)

The fusion protein of claim 21, further comprising at least one nuclear localization signal, at least one cell-penetrating domain, at least one marker domain, or a combination thereof.

### Claim 25 (depends on 21)

The fusion protein of claim 21, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120.

### Claim 26 (depends on 21)

A system for eukaryotic genome modification comprising the fusion protein of claim 21 and an engineered guide RNA, wherein the engineered guide RNA comprises the nucleotide sequence of SEQ ID NO:31.