# US 11,310,179 B2 · RNA targeting methods and compositions

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- Number: US 11,310,179 B2
- Document ID: US11310179B2
- Status: Granted
- Kind: B2
- Application number: 17/352,551
- Filed: 2021-06-21
- Published: 2021-11-04
- Granted: 2022-04-19
- Earliest claimed: 2017-08-22
- Assignees: The Salk Institute for Biological Studies
- Applicants: Salk Institute for Biological Studies
- Inventors: Patrick D. Hsu; Silvana Konermann
- Web page: https://patentgrep.com/patent/US11310179B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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 15/8201: {Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/85: for animal cells
- H04L 9/08: Key distribution {or management, e.g. generation, sharing or updating, of cryptographic keys or passwords (network architectures or network communication protocols for supporting key management in a packet data network H04L63/06)}
- H04L 9/30: Public key, i.e. encryption algorithm being computationally infeasible to invert or user's encryption keys not requiring secrecy
- H04L 51/08: Annexed information, e.g. attachments
- H04L 51/18: Commands or executable codes
- H04L 51/212: using filtering or selective blocking
- H04L 51/214: using selective forwarding
- C12N 2310/16: Aptamers
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3519: Fusion with another nucleic acid
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- H04L: TRANSMISSION OF DIGITAL INFORMATION, e.g. TELEGRAPHIC COMMUNICATION (arrangements common to telegraphic and telephonic communication H04M)

## Abstract

Provided herein are CRISPR/Cas methods and compositions for targeting RNA molecules, which can be used to detect, edit, or modify a target RNA.

## Claims

### Claim 1

A non-naturally occurring or engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system for targeting one or more target RNA molecules, comprising:
(a) at least one Cas13d protein comprising at least 95% sequence identity to SEQ ID NO: 212, or a nucleic acid molecule encoding the at least one Cas13d protein; and
(b) at least one CRISPR-Cas system guide RNA (gRNA) comprising one or more direct repeat (DR) sequences and one or more spacer sequences, which hybridizes with the one or more target RNA molecules, or at least one nucleic acid molecule encoding the gRNA,
whereby the Cas13d protein forms a complex with the gRNA, wherein the gRNA directs the complex to the one or more target RNA molecules and targets the one or more target RNA molecules.

### Claim 2 (depends on 1)

An isolated cell comprising the system of claim 1.

### Claim 3 (depends on 1)

A ribonucleoprotein complex comprising the system of claim 1, wherein the ribonucleoprotein complex comprises the (a) at least one Cas13d protein comprising at least 95% sequence identity to SEQ ID NO: 212 and the (b) at least one CRISPR-Cas system gRNA comprising one or more DR sequences and one or more spacer sequences, which hybridizes with the one or more target RNA molecules,
wherein the gRNA directs the ribonucleoprotein complex to the one or more target RNA molecules and targets the one or more target RNA molecules.

### Claim 4 (depends on 1)

The system of claim 1, wherein targeting one or more target RNA molecules comprises one or more of cutting the one or more target RNA molecules, nicking the one or more target RNA molecules, increasing expression of the one or more target RNA molecules as compared to an amount of target RNA in a corresponding cell or sample not contacted with the at least one Cas13d protein, decreasing expression of the one or more target RNA molecules as compared to an amount of target RNA in a corresponding cell or sample not contacted with the at least one Cas13d protein, visualizing or detecting the one or more target RNA molecules, labeling the one or more target RNA molecules, capturing molecules bound or tethered to the one or more target RNA molecules, enriching the one or more target RNA molecules, depleting the one or more target RNA molecules, editing the one or more target RNA molecules, trafficking the one or more target RNA molecules, splicing or perturbing the splicing of the one or more target RNA molecules, and masking the one or more target RNA molecules.

### Claim 5 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein comprises at least 96% sequence identity to SEQ ID NO: 212.

### Claim 6 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein comprises at least 97% sequence identity to SEQ ID NO: 212.

### Claim 7 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein comprises at least 98% sequence identity to SEQ ID NO: 212.

### Claim 8 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein comprises at least 99% sequence identity to SEQ ID NO: 212.

### Claim 9 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein comprises SEQ ID NO: 212.

### Claim 10 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein further comprises one or more subcellular localization signals, effector domains, purification tags, affinity tags, or combinations thereof.

### Claim 11 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising at least 95% sequence identity to SEQ ID NO: 213.

### Claim 12 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising at least 96% sequence identity to SEQ ID NO: 213.

### Claim 13 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising at least 97% sequence identity to SEQ ID NO: 213.

### Claim 14 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising at least 98% sequence identity to SEQ ID NO: 213.

### Claim 15 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising at least 99% sequence identity to SEQ ID NO: 213.

### Claim 16 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises one or more DR sequences comprising SEQ ID NO: 213.

### Claim 17 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises a nucleic acid aptamer.

### Claim 18 (depends on 17)

The system of claim 17 wherein the nucleic acid aptamer inserted into the gRNA comprises MS2, PP7, or Qβ.

### Claim 19 (depends on 1)

The system of claim 1, wherein the at least one gRNA comprises modified nucleotides.

### Claim 20 (depends on 2)

The isolated cell of claim 2, wherein the cell is a eukaryotic cell or a bacterial cell.

### Claim 21 (depends on 20)

The isolated cell of claim 20, wherein the eukaryotic cell is a mammalian cell.

### Claim 22 (depends on 20)

The isolated cell of claim 20, wherein the eukaryotic cell is a plant cell.

### Claim 23 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein further comprises a nuclear localization signal (NLS).

### Claim 24 (depends on 23)

The system of claim 23, wherein the NLS comprises SEQ ID NO: 256 or SEQ ID NO: 258.

### Claim 25 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein further comprises a nuclear export signal (NES).

### Claim 26 (depends on 25)

The system of claim 25, wherein the NES comprises an adenovirus type 5 E1B nuclear export sequence, an HIV nuclear export sequence, a MAPK nuclear export sequence, or a PTK2 nuclear export sequence.

### Claim 27 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein further comprises an effector domain that can cleave RNA, edit a nucleotide, edit a ribonucleotide, methylate RNA, or demethylate RNA.

### Claim 28 (depends on 1)

The system of claim 1, wherein the at least one Cas13d protein is catalytically inactive for ribonuclease activity.

### Claim 29 (depends on 28)

The system of claim 28, wherein the catalytically inactive Cas13d protein comprises one or more mutated HEPN domains.

### Claim 30 (depends on 28)

The system of claim 28, wherein the catalytically inactive Cas13d protein comprises a mutated HEPN1 domain or a mutated HEPN2 domain.