# US 11,439,712 B2 · Methods and compositions for RNA-directed repression of transcription using CRISPR-associated genes

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- Number: US 11,439,712 B2
- Document ID: US11439712B2
- Status: Granted
- Kind: B2
- Application number: 15/302,655
- Filed: 2015-04-07
- Published: 2017-02-02
- Granted: 2022-09-13
- Earliest claimed: 2014-04-08
- Assignees: North Carolina State University
- Applicants: North Carolina State University
- Inventors: Chase Beisel; Michelle Luo
- Web page: https://patentgrep.com/patent/US11439712B2

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/72: Expression systems using regulatory sequences derived from the lac-operon
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/30: Special therapeutic applications
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention is directed to methods and compositions for targeted gene silencing that provide the ability to not only repress expression but to modulate the repression of expression of one or more target genes. In one aspect, a recombinant nucleic acid molecule is provided comprising a nucleotide sequence encoding a subset of CRISPR-cas polypeptides, or functional fragments thereof, from a type-I CRISPR-cas system. In some aspects, a recombinant nucleic acid of the invention comprises a nucleotide sequence encoding three or more Type I Cascade polypeptides having substantial identity to a type I Cascade polypeptide.

## Claims

### Claim 1

A method for repressing expression of a first target gene in a bacterium or an archaeon, the method comprising introducing into the bacterium or the archaeon:
(a) a first nucleic acid sequence encoding at least three polypeptides of a Type I CASCADE, wherein the Type I CASCADE is a Type I-B CASCADE, a Type I-C CASCADE, a Type I-E CASCADE, a Type I-F CASCADE, a Type I-A CASCADE or a Type I-D CASCADE; and
(b) a second nucleic acid sequence encoding at least one CRISPR array comprising two or more repeat sequences and at least one spacer sequence that is complementary to a first target sequence from the first target gene, wherein the at least one spacer sequence is increased in length by about 1 to about 100 nucleotides when compared to a normal spacer length, wherein:
the Type I-B CASCADE comprises a Cas6b polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:18-20 or 335-340, a Cas8b (Csh1) polypeptide having at least 95% sequence identity to any one of the sequence of SEQ ID NOs:21, 22, or 1043-1158, a Cas7 (Csh2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:23, 24, or 1159-1209, and a Cas5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:25, 26, or 1210-2372,
the Type I-C CASCADE comprises a Cas5d polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:27, 28, or 2373-2973, a Cas8c (Csd1) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:29, 30, or 2974-3847, and a Cas7 (Csd2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:31, 32, or 3848-4371,
the Type I-E CASCADE comprises a Cse1 (CasA) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:41, 42, or 5235-6111, a Cse2 (CasB) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:43, 44, or 6112-6823, a Cas7 (CasC) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:45, 46, or 6824-7032, a Cas5 (CasD) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:47, 48, or 7033-7251, and a Cas6e (CasE) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:49, 50, or 7252-7520,
the Type I-F CASCADE comprises a Cys1 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:51, 52, or 7521-8614, a Cys2 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:53, 54, or 8614-9867, a Cas7 (Cys3) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:55, 56, or 9868-11057, and a Cas6f polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:57, 58, or 11058-11528,
the Type I-A CASCADE comprises a Cas7 (Csa2) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:1-3, a Cas8a1 (Csx13) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:4, or 341-640 or a Cas8a2 (Csx9) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:5, 6, or 641-666, a Cas5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:7-9, or 667-871, a Csa5 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:872-1042, a Cas6a polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:10, 11, or 325-328, a Cas3′ polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:12-14, or 329-331, and a Cas3″ polypeptide having no nuclease activity and having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:15-17, or 332-334, said polypeptides having at least 95% sequence identity to one of the sequences of SEQ ID NOs:1-17, 325-334, or 341-1042, and
the Type I-D CASCADE comprises a Cas10d (Csc3) polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:33, 34, or 4372-4678, a Csc2 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:35, 36, or 4679-4985, Csc1 polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:37, 38, or 4986-5234, a Cas6d polypeptide having at least 95% sequence identity to any one of the sequences of SEQ ID NOs:39 or 40, thereby repressing expression of the first target gene in the bacterium or archaeon.

### Claim 2 (depends on 1)

The method of claim 1, wherein repressing expression of the target gene comprises an increase in repression of the target gene.

### Claim 3 (depends on 1)

The method of claim 1, wherein the CRISPR array is operably linked to a promoter and/or a terminator.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first nucleic acid sequence is operably linked to a promoter, regulatory element, or any combination thereof.

### Claim 5 (depends on 1)

The method of claim 1, wherein the at least one CRISPR array comprises at least two spacer sequences, a first spacer sequence and a second spacer sequence, the first spacer sequence being complementary to a first target site in the first target gene and the second spacer sequence being complementary to a second target site in the first target gene, thereby modulating expression of the first target gene.

### Claim 6 (depends on 1)

The method of claim 1, wherein the at least one CRISPR array comprises at least two spacer sequences, a first spacer sequence and a second spacer sequence, the first spacer sequence being complementary to the first target gene and the second spacer being complementary to a second target sequence in a second target gene, thereby modulating expression of at least two genes in the bacterium or the archaeon.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first target sequence comprises all or a part of a promoter sequence.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first target sequence is located on a coding strand of a transcribed region of the target gene.

### Claim 9 (depends on 6)

The method of claim 6, wherein the second target sequence comprises all or a part of a promoter sequence.

### Claim 10 (depends on 6)

The method of claim 6, wherein the second target sequence is located on a coding strand of a transcribed region of the target gene.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are on the same or a different expression cassettes or vectors.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked together on the same expression cassette or vector.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are transiently or stably incorporated into the bacterium or the archaeon.

### Claim 14 (depends on 1)

The method of claim 1, wherein the two or more repeat sequences comprise a first repeat sequence and a second repeat sequence and the first repeat sequence is linked to the 5′ end of the spacer sequence and the second repeat sequence is linked to the 3′ end of the spacer sequence and the first repeat sequence or the second repeat sequence comprises a portion of a wild type repeat sequence having at least three contiguous nucleotides of a wild type repeat sequence.

### Claim 15 (depends on 1)

The method of claim 1, wherein the bacterium is pathogenic.

### Claim 16 (depends on 1)

The method of claim 1, wherein the at least one spacer sequence comprises a length of about 32 nucleotides to about 100 nucleotides.

### Claim 17 (depends on 1)

The method of claim 1, wherein the at least one spacer sequence is increased in length by 6, 12, 18, 24, 30 or 36 nucleotides when compared to a normal spacer length.