# US 12,110,490 B2 · CRISPR enzymes and systems

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- Number: US 12,110,490 B2
- Document ID: US12110490B2
- Status: Granted
- Kind: B2
- Application number: 16/063,643
- Filed: 2016-12-16
- Published: 2019-08-01
- Granted: 2024-10-08
- Earliest claimed: 2015-12-18
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology; President and Fellows of Harvard College
- Inventors: Feng Zhang; Bernd Zetsche; Fei Ran; James E. Dahlman
- Web page: https://patentgrep.com/patent/US12110490B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/902: {using homologous recombination}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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 composition comprising:
a non-naturally occurring or engineered guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the gRNA is capable of forming a complex with a Cpf1 effector protein, wherein the gRNA comprises a loop comprising an aptamer sequence that is capable of binding to an adaptor protein; and
a mutated Cpf1 effector protein, wherein the mutated Cpf1 effector protein comprises at least one mutation, such that the mutated Cpf1 effector protein has no more than 5% of the nuclease activity of a corresponding wild-type Cpf1 effector protein not having the at least one mutation, and wherein the mutated Cpf1 effector protein comprises one or more nuclear localization sequences.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the composition further comprises a fusion protein comprising an adaptor protein fused to one or more heterologous functional domains.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the mutated Cpf1 effector protein has a diminished nuclease activity of at least 97%, or 100% as compared with the corresponding wild-type Cpf1 effector protein not having the at least one mutation.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the mutated Cpf1 effector protein comprises at least one mutation selected from D908A, E993A, or D1263A according to AsCpf1 protein or a corresponding mutation in an ortholog.

### Claim 5 (depends on 2)

The composition of claim 2, wherein the mutated Cpf1 effector protein comprises at least one mutation selected from D832A, E925A, D947A or D1180A according to LbCpf1 protein or a corresponding mutation in an ortholog.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the mutated Cpf1 effector protein comprises two or more mutations.

### Claim 7 (depends on 2)

The composition of claim 2, wherein the mutated Cpf1 effector protein is fused to one or more heterologous functional domains.

### Claim 8 (depends on 2)

The composition of claim 2, wherein the fusion protein comprises a linker between the adaptor protein and the heterologous functional domain, the linker optionally comprising a GlySer linker.

### Claim 9 (depends on 2)

The composition of claim 2, wherein the one or more heterologous functional domains fused to the adaptor protein comprise a transcriptional activation domain.

### Claim 10 (depends on 7)

The composition of claim 7, wherein the one or more heterologous functional domains fused to the mutated Cpf1 effector protein comprise a transcriptional activation domain.

### Claim 11 (depends on 9)

The composition of claim 9, wherein the transcriptional activation domain is VP64, p65, MyoD1, HSF1, RTA or SET7/9.

### Claim 12 (depends on 10)

The composition of claim 10, wherein the transcriptional activation domain is VP64, p65, MyoD1, HSF1, RTA or SET7/9.

### Claim 13 (depends on 2)

The composition of claim 2, wherein the one or more heterologous functional domains fused to the adaptor protein comprise a transcriptional repressor domain.

### Claim 14 (depends on 7)

The composition of claim 7, wherein the one or more heterologous functional domains fused to the mutated Cpf1 effector protein comprise a transcriptional repressor domain.

### Claim 15 (depends on 13)

The composition of claim 13, wherein the transcriptional repressor domain is a KRAB domain.

### Claim 16 (depends on 13)

The composition of claim 13, wherein the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or a SID4X domain.

### Claim 17 (depends on 2)

The composition of claim 2, wherein at least one of the one or more heterologous functional domains fused to the adaptor protein has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity.

### Claim 18 (depends on 7)

The composition of claim 7, wherein at least one of the one or more heterologous functional domains fused to the mutated Cpf1 effector protein has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity or chemical inducibility or light inducibility.

### Claim 19 (depends on 2)

The composition of claim 2, wherein the adaptor protein is a bacteriophage coat protein selected from the group consisting of MS2, PP7, Qβ, GA, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, and PRR1.

### Claim 20 (depends on 2)

The composition of claim 2, wherein the cell is a eukaryotic cell.

### Claim 21 (depends on 20)

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

### Claim 22 (depends on 21)

The composition of claim 21, wherein the mammalian cell is a human cell.

### Claim 23 (depends on 20)

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

### Claim 24 (depends on 2)

A non-human transgenic cell or cell line which has been transformed with the composition according to claim 2.

### Claim 25 (depends on 2)

A non-human transgenic mammalian model, wherein the model has been transformed with the composition according to claim 2.

### Claim 26 (depends on 2)

An isolated host cell or cell line comprising the composition of claim 2.

### Claim 27 (depends on 26)

The isolated host cell or cell line according to claim 26, wherein the cell is a eukaryotic cell.

### Claim 28 (depends on 26)

The isolated host cell or cell line according to claim 26, wherein the cell is an animal cell.

### Claim 29 (depends on 28)

The isolated host cell or cell line according to claim 28, wherein the cell is a human cell.

### Claim 30 (depends on 26)

The isolated host cell or cell line according to claim 26, comprising a stem cell or stem cell line.

### Claim 31 (depends on 26)

The isolated host cell or cell line according to claim 26, wherein the cell is a plant cell.

### Claim 32

A vector system comprising one or more vector(s) comprising nucleic acid molecule(s) encoding: a guide RNA (gRNA) and a mutated Cpf1 effector protein,
wherein the mutated Cpf1 effector protein comprises at least one mutation, such that the mutated Cpf1 effector protein has no more than 5% of the nuclease activity of a corresponding wild-type Cpf1 effector protein not having the at least one mutation, and wherein the mutated Cpf1 effector protein comprises at least one or more nuclear localization sequences, and
wherein the gRNA is a non-naturally occurring or engineered gRNA comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the gRNA is capable of forming a complex with a Cpf1 effector protein, wherein the gRNA comprises a loop comprising an aptamer sequence that is capable of binding to an adaptor protein.

### Claim 33 (depends on 32)

The vector system of claim 32, nucleic acid molecule(s) further encoding a fusion protein comprising an adaptor protein fused to one or more heterologous functional domains.