# US 2019/0169595 A1 · Rna targeting methods and compositions

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- Number: US 2019/0169595 A1
- Document ID: US20190169595A1
- Status: Published application
- Kind: A1
- Application number: 16/257,493
- Filed: 2019-01-25
- Published: 2019-06-06
- 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/US20190169595A1

## 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 method of targeting one or more target RNA molecules, comprising:
contacting one or more target RNA molecules with a non-naturally occurring or engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system comprising:
at least one Cas13d protein, or a nucleic acid molecule encoding the at least one Cas13d protein; and
at least one CRISPR-Cas system guide RNA (gRNA) that 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

A CRISPR-Cas system-mediated RNA targeting method, comprising:
contacting one or more target RNA molecules with a non-naturally occurring or engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) system comprising:
at least one Cas13d protein, or a nucleic acid molecule encoding the at least one Cas13d protein; and
at least one CRISPR-Cas system gRNA that 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 3 (depends on 1)

The method of claim 1, wherein contacting the one or more target RNA molecules with the non-naturally occurring or engineered CRISPR-Cas system comprises introducing into a cell containing the one or more target RNA molecules the non-naturally occurring or engineered CRISPR-Cas system.

### Claim 4 (depends on 1)

The method of claim 1, wherein the one or more target RNA molecules or the cell are contacted with a complex comprising the at least one Cas13d protein and the at least one CRISPR-Cas system gRNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the least one Cas13d protein:
comprises one or more HEPN domains,
is no more than 150 kD, no more than 140 kd, no more than 130 kD, no more than 120 kD, such as about 90 to 120 kD, about 100 to 120 kD or about 110 kD,
comprises one or more mutated HEPN domains, and can process the guide RNA, but cannot cut the one or more target RNA molecules,
comprises an Cas13d ortholog from a prokaryotic genome or metagenome, gut metagenome, an activated sludge metagenome, an anaerobic digester metagenome, a chicken gut metagenome, a human gut metagenome, a pig gut metagenome, a bovine gut metagenome, a sheep gut metagenome, a goat gut metagenome, a capybara gut metagenome, a primate gut metagenome, a termite gut metagenome, a fecal metagenome, a genome from the Order Clostridiales, or the Family Ruminococcaceae,
comprises an Cas13d ortholog from Ruminococcus albus, Eubacterium siraeum, Ruminococcus flavefaciens strain XPD3002, Ruminococcus flavefaciens FD-1, uncultured Eubacterium sp TS28-c4095, uncultured Ruminococcus sp., Ruminococcus bicirculans, or Ruminococcus sp CAG57,
comprises at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 138, 147, 149, 153, 155, 158, 160, 162, 164, 166, 168, 170, 175, 177, 179, 181, 183, 185, 187, 194, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, or 253,
comprises a motif shown in SEQ ID NO: 195, 196 or 197;
comprises one or more amino acid deletions;
comprises one or more amino acid insertions;
is divided into 2 or more fragments that can reconstitute a full-length functional enzyme when co-delivered;
is modified to be a chimera of different Cas13d orthologs;
is truncated by at least 1%, such as 1 to 10%;
optionally further comprises one or more subcellular localization signals, effector domains, purification tags, affinity tags, or
combinations thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the vector encoding the at least one Cas13d protein comprises:
at least one Cas13d protein coding sequence that is codon optimized for expression in a eukaryotic cell;
at least one Cas13d protein coding sequence that is codon optimized for expression in a prokaryotic cell,
at least one Cas13d protein coding sequence codon optimized for expression in a human cell and comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 142, 143, 144, or 145;
at least one Cas13d protein coding sequence comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124, 125, 126, 127, 128, 139, 140 or 141;
at least one Cas13d protein coding sequence encoding a protein comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 138, 147, 149, 153, 155, 158, 160, 162, 164, 166, 168, 170, 175, 177, 179, 181, 183, 185, 187, 194, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, or 253;
at least one Cas13d ortholog from a prokaryotic genome or metagenome; or
at least on Cas13d ortholog from a gut metagenome, an activated sludge metagenome, an anaerobic digester metagenome, a chicken gut metagenome, a human gut metagenome, a pig gut metagenome, a bovine gut metagenome, a sheep gut metagenome, a goat gut metagenome, a capybara gut metagenome, a primate gut metagenome, a termite gut metagenome, a fecal metagenome, a genome from the Order Clostridiales, or the Family Ruminococcaceae.

### Claim 7 (depends on 1)

The method of claim 1, wherein gRNA that hybridizes with the one or more target RNA molecules comprises:
a mature gRNA,
a pre-gRNA,
one or more direct repeat (DR) sequences, wherein the DR sequence can be a full length DR or a truncated DR (such as truncated by at least 1 nt, at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, such as 1-3 nt, 3-6 nt, or 5-10 nt),
one or more spacer sequences,
one or more sequences comprising DR-spacer-DR-spacer,
one or more DR sequences comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 148, 150, 151, 152, 154, 156, 157, 159, 161, 163, 165, 167, 169, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, or 254,
optionally a nucleic acid aptamer inserted into the gRNA (e.g., MS2 stem loops),
or combinations thereof.

### Claim 8 (depends on 1)

The method 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, activating the one or more target RNA molecules, deactivating the one or more target RNA molecules, visualizing or detecting the one or more target RNA molecules, labeling the one or more target RNA molecules, binding 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 9 (depends on 1)

The method of claim 1, wherein the method treats a disease, and wherein the one or more target RNA molecules is associated with the disease.

### Claim 10 (depends on 1)

The method of claim 1, wherein the method is a diagnostic method wherein
contacting the one or more target RNA molecules with the non-naturally occurring or engineered CRISPR-Cas system, comprises contacting the non-naturally occurring or engineered CRISPR-Cas system with a sample comprising the one or more target RNA molecules and with a separate RNA comprising a label,
optionally transcribing nucleic acid molecules present in the sample,
optionally amplifying nucleic acid molecules present in the sample, and
detecting the label, wherein detecting the label indicates the presence of one or more target RNAs in the sample.

### Claim 11

An isolated protein, comprising:
at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113;
a motif shown in SEQ ID NO: 195, 196 or 197,
an Cas13d ortholog from a prokaryotic genome or metagenome, gut metagenome, an activated sludge metagenome, an anaerobic digester metagenome, a chicken gut metagenome, a human gut metagenome, a pig gut metagenome, a bovine gut metagenome, a sheep gut metagenome, a goat gut metagenome, a capybara gut metagenome, a primate gut metagenome, a termite gut metagenome, a fecal metagenome, a genome from the Order Clostridiales, or the Family Ruminococcaceae, or
combinations thereof.

### Claim 12 (depends on 11)

The isolated protein of claim 11, further comprising a subcellular localization signal.

### Claim 13 (depends on 11)

The isolated protein of claim 11, wherein the protein comprises a mutation in at least one native HEPN domain.

### Claim 14

An isolated guide RNA (gRNA), comprising:
one or more direct repeat (DR) sequences comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 148, 150, 151, 152, 154, 156, 157, 159, 161, 163, 165, 167, 169, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, or 254, or a truncated version thereof.

### Claim 15 (depends on 14)

The isolated gRNA of claim 14, further comprising one or more spacer sequences specific for a target RNA.

### Claim 16 (depends on 11)

A ribonucleoprotein (RNP) complex comprising:
the isolated protein of claim 11, and
an isolated gRNA comprising one or more direct repeat (DR) sequences comprising at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 129, 130, 131, 132, 133, 134, 135, 136, 137, 148, 150, 151, 152, 154, 156, 157, 159, 161, 163, 165, 167, 169, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, or 254, 254, or a truncated version thereof.

### Claim 17 (depends on 11)

An isolated nucleic acid molecule encoding the protein of claim 11.

### Claim 18 (depends on 17)

The isolated nucleic acid molecule of claim 17, comprising:
at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124, 125, 126, 127, 128, 139, 140 or 141;
at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 142, 143, 144, or 145; or
encoding for a protein sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113.

### Claim 19 (depends on 11)

A composition, comprising the isolated protein of claim 11, and a pharmaceutically acceptable carrier.

### Claim 20 (depends on 11)

A kit, comprising:
the isolated protein of claim 11, and
a delivery system, a label, or both.