# US 12,215,318 B2 · Crispr enzymes and systems

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

- Number: US 12,215,318 B2
- Document ID: US12215318B2
- Status: Granted
- Kind: B2
- Application number: 15/960,064
- Filed: 2018-04-23
- Published: 2019-03-28
- Granted: 2025-02-04
- Earliest claimed: 2015-10-22
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Inventors: Feng Zhang; Aaron Smargon; Neena Pyzocha; David Benjamin Turitz Cox; Eric S. Lander
- Web page: https://patentgrep.com/patent/US12215318B2

## CPC

- 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 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- 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 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16B 30/20: Sequence assembly
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12Q 2600/156: Polymorphic or mutational markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

## Claims

### Claim 1

A non-naturally occurring or engineered composition consisting of
i) a Type VI-B CRISPR-Cas effector protein, and
ii) a Type VI-B CRISPR-Cas recombinant guide RNA,
wherein the recombinant guide RNA comprises, from 5′ to 3′ orientation, a) a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence,
whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence, and wherein the Type VI-B CRISPR-Cas effector protein and Type VI-B CRISPR-Cas guide RNA do not naturally occur together.

### Claim 2 (depends on 1)

The non-naturally occurring or engineered composition of claim 1,
wherein the guide sequence hybridizes to a target RNA sequence in a prokaryotic cell, or hybridizes to a target RNA sequence in a eukaryotic cell.

### Claim 3 (depends on 1)

The non-naturally occurring or engineered composition of claim 1, wherein the Type VI-B CRISPR-Cas effector protein is selected from Type VI-B CRISPR-Cas effector proteins obtained from Paludibacter propionicigenes WB4, Prevotella sp. P5-60, Prevotella sp. P4-76, Prevotella sp. P5-125, Prevotella sp. P5-119, Capnocytophaga canimorsus Cc5, Phaeodactylibacter xiamenensis, Porphyromonas gingivalis W83, Porphyromonas gingivalis F0570, Porphyromonas gingivalis ATCC 33277, Porphyromonas gingivalis F0185, Porphyromonas gingivalis F0185, Porphyromonas gingivalis SJD2, Porphyromonas gingivalis F0568, Porphyromonas gingivalis W4087, Porphyromonas gingivalis W4087, Porphyromonas gingivalis F0568, Porphyromonas gingivalis, Porphyromonas gulae, Bacteroides pyogenes F0041, Bacteroides pyogenes JCM 10003, Alistipes sp. ZOR0009, Flavobacterium branchiophilum FL-15, Prevotella sp. MA2016, Myroides odoratimimus CCUG 10230, Myroides odoratimimus CCUG 3837, Myroides odoratimimus CCUG 3837, Myroides odoratimimus CCUG 12901, Myroides odoratimimus CCUG 12901, Myroides odoratimimus, Bergeyella zoohelcum ATCC 43767, Capnocytophaga cynodegmi, Bergeyella zoohelcum ATCC 43767, Flavobacterium sp. 316, Psychroflexus torquis ATCC 700755, Flavobacterium columnare ATCC 49512, Flavobacterium columnare, Flavobacterium columnare, Flavobacterium columnare, Chryseobacterium sp. YR477, Riemerella anatipestifer ATCC 11845=DSM 15868, Riemerella anatipestifer RA-CH-2, Riemerella anatipestifer, Riemerella anatipestifer, Riemerella anatipestifer, Prevotella saccharolytica F0055, Prevotella saccharolytica JCM 17484, Prevotella buccae ATCC 33574, Prevotella buccae ATCC 33574, Prevotella buccae D17, Prevotella sp. MSX73, Prevotella pallens ATCC 700821, Prevotella pallens ATCC 700821, Prevotella intermedia ATCC 25611=DSM 20706, Prevotella intermedia, Prevotella intermedia 17, Prevotella intermedia, Prevotella intermedia, Prevotella intermedia ZT, Prevotella aurantiaca JCM 15754, Prevotella pleuritidis F0068, Prevotella pleuritidis JCM 14110, Prevotella falsenii DSM 22864=JCM 15124, Porphyromonas gulae, Porphyromonas sp. COT-052 OH4946, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gulae, Porphyromonas gingivalis TDC60, Porphyromonas gingivalis ATCC 33277, Porphyromonas gingivalis A7A1-28, Porphyromonas gingivalis JCVI SC001, Porphyromonas gingivalis W50, Porphyromonas gingivalis, Porphyromonas gingivalis AJW4, Porphyromonas gingivalis, and Porphyromonas gingivalis.

### Claim 4 (depends on 1)

A Type VI-B CRISPR-Cas vector system for providing the composition of claim 1, which comprises one or more vectors comprising: a first regulatory element operably linked to a nucleotide sequence encoding the Type VI-B CRISPR-Cas effector protein, and a second regulatory element operably linked to a nucleotide sequence encoding the Type VI-B CRISPR-Cas recombinant guide RNA.

### Claim 5 (depends on 4)

The vector system of claim 4, which further comprises: a regulatory element operably linked to a nucleotide sequence of a Type VI-B CRISPR-Cas accessory protein.

### Claim 6 (depends on 5)

The vector system of claim 5, wherein the nucleotide sequences encoding the Type VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Cas accessory protein are codon optimized for expression in a eukaryotic cell.

### Claim 7 (depends on 4)

The vector system of claim 4, wherein the nucleotide sequence encoding a Type VI-B CRISPR-Cas effector protein is codon optimized for expression in a eukaryotic cell.

### Claim 8 (depends on 4)

The vector system of claim 4, which is a single vector.

### Claim 9 (depends on 4)

The vector system of claim 4, wherein the one or more vectors comprise are viral vectors.

### Claim 10 (depends on 4)

The vector system of claim 4, wherein the one or more vectors are one or more retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.

### Claim 11 (depends on 1)

A delivery system configured to deliver the composition of claim 1,
wherein the Type VI-B CRISPR-Cas system effector protein forms a complex with the guide RNA,
wherein the heterologous guide sequence directs sequence-specific binding to the target RNA sequence,
whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence.

### Claim 12 (depends on 11)

The delivery system of claim 11, which comprises one or more vectors or one or more polynucleotide molecules, the one or more vectors or polynucleotide molecules comprising one or more polynucleotide molecules encoding the Type VI-B CRISPR-Cas effector protein and one or more nucleic acid components of the non-naturally occurring or engineered composition.

### Claim 13 (depends on 11)

The delivery system of claim 11, which comprises a delivery vehicle selected from the group consisting of liposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun and one or more viral vector(s).

### Claim 14 (depends on 1)

The composition of claim 1, wherein the Type VI-B CRISPR-Cas effector protein is from a micro-organism selected from Bergeyella zoohelcum and Prevotella buccae.

### Claim 15

A non-naturally occurring or engineered composition consisting of
i) a Type VI-B CRISPR-Cas effector protein,
ii) a Type VI-B CRISPR-Cas recombinant guide RNA, and
iii) a Type VI-B CRISPR-Cas accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity, or a Type VI-B CRISPR-Cas accessory protein that represses Type VI-B CRISPR-Cas effector protein activity,
wherein the recombinant guide RNA comprises, from 5′ to 3′ orientation, a) a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence, whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence, and wherein the Type VI-B CRISPR-Cas effector protein and Type VI-B CRISPR-Cas guide RNA do not naturally occur together.

### Claim 16 (depends on 15)

The non-naturally occurring or engineered composition of claim 15, wherein the accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity is a csx28 protein, and the accessory protein that represses Type VI-B CRISPR-Cas effector protein activity is csx27 protein.

### Claim 17 (depends on 15)

The non-naturally occurring or engineered composition of claim 15, wherein the Type VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Cas accessory protein are from the same organism or a different organism.

### Claim 18 (depends on 15)

A method of modifying expression of a target RNA sequence comprising contacting a target RNA sequence with
the composition of claim 15,
wherein the Type VI-B CRISPR-Cas system effector protein forms a complex with the guide RNA,
wherein the heterologous guide sequence directs sequence-specific binding to the target RNA sequence in a cell,
whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the heterologous guide sequence that is hybridized to the target RNA sequence, and
whereby expression of the target RNA sequence is modified.

### Claim 19 (depends on 18)

The method of claim 18, further comprising contacting the target RNA sequence with the Type VI-B CRISPR-Cas accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity, or contacting the target RNA sequence with the Type VI-B CRISPR-Cas accessory protein that represses Type VI-B CRISP-Cas effector activity.

### Claim 20 (depends on 19)

The method of claim 19, wherein the accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity is a csx28 protein, and wherein the accessory protein that represses Type VI B CRISPR-Cas effector protein activity is a csx27 protein.

### Claim 21 (depends on 18)

The method of claim 18, wherein modifying expression of the target gene comprises cleaving the target RNA, or increasing or decreasing expression of the target RNA.

### Claim 22 (depends on 18)

The method of claim 18, wherein the target gene is in a prokaryotic cell, or in a eukaryotic cell.

### Claim 23

A non-naturally occurring or engineered composition consisting of
i) a Type VI-B CRISPR-Cas effector protein having one or more nuclear localization signals and one or more functional domains covalently associated with the Type VI-B CRISPR-Cas effector protein, and
ii) a Type VI-B CRISPR-Cas recombinant guide RNA,
wherein the recombinant guide RNA comprises, from 5′ to 3′ orientation, a) a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence, whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence, and wherein the Type VI-B CRISPR-Cas effector protein and Type VI-B CRISPR-Cas guide RNA do not naturally occur together.

### Claim 24 (depends on 23)

The non-naturally occurring or engineered composition of claim 23, wherein the one or more functional domains cleave the target RNA sequence.

### Claim 25 (depends on 23)

The non-naturally occurring or engineered composition of claim 23, wherein the one or more functional domains modify transcription or translation of the target RNA sequence.

### Claim 26 (depends on 23)

The composition of claim 23, wherein the Type VI-B CRISPR-Cas effector protein contains one or more mutations within a HEPN domain, whereby the complex can deliver to a target RNA sequence in a prokaryotic cell or eukaryotic cell an epigenetic modifier, or a transcriptional or translational activation or repression signal.