CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-10648020-B2
Application number	US-201715844608-A
Country	US
Kind code	B2
Filing date	Dec 17, 2017
Priority date	Jun 18, 2015
Publication date	May 12, 2020
Grant date	May 12, 2020

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.

First claim

What is claimed:

1. A method for detecting a target DNA in a eukaryotic nucleic acid sample, comprising:
(a) contacting the nucleic acid sample with a CRISPR-Cpf1 complex comprising a Cpf1 effector protein and a guide polynucleotide that comprises a guide sequence linked to a direct repeat sequence, wherein the guide sequence directs sequence-specific binding of the CRISPR-Cpf1 complex to the target DNA, and wherein the complex lacks a tracr sequence; and
(b) detecting presence of the target DNA by detecting occurrence of binding of the CRISPR-Cpf1 complex to the target DNA or cleavage of the target DNA by the CRISPR-Cpf1 complex.

2. The method of claim 1 , wherein the Cpf1 effector protein is of a bacterial species selected from the group consisting of Francisella tularensis 1 , Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1 , Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10 , Parcubacteria bacterium GW2011_GWC2_44_17 , Smithella sp. SCADC, Acidaminococcus sp. BV3L6 , Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237 , Leptospira inadai, Lachnospiraceae bacterium ND2006 , Porphyromonas crevioricanis 3 , Prevotella disiens and Porphyromonas macacae.

3. The method of claim 1 , wherein the Cpf1 effector protein is Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), or Franscisella novicida U112 Cpf1 (FnCpf1).

4. The method of claim 3 , wherein the Cpf1 effector protein is Franscisella novicida U112 Cpf1.

5. The method of claim 3 , wherein the Cpf1 effector protein is Acidaminococcus sp. BV3L6 Cpf1.

6. The method of claim 3 , wherein the Cpf1 effector protein is Lachnospiraceae bacterium ND2006 Cpf1.

7. The method of claim 1 , wherein the occurrence of binding of the CRISPR-Cpf1 complex to the target DNA or cleavage of the target DNA by the CRISPR-Cpf1 complex is detected by a fluorescent signal.

8. The method of claim 1 , wherein the target DNA is associated with a disease.

9. The method of claim 1 , wherein the target DNA is associated with cancer.

10. The method of claim 1 , wherein the target DNA is associated with a virus.

11. The method of claim 1 , wherein the target DNA comprises a target sequence hybridizable with the guide sequence, wherein the target sequence is located at 3′ of a Protospacer Adjacent Motif (PAM).

12. The method of claim 11 , wherein the PAM comprises a 5′ T-rich motif.

13. The method of claim 11 , wherein the PAM sequence is TTN, where N is A/C/G or T and the effector protein is FnCpf1, or wherein the PAM sequence is TTTV, where V is A/C or G and the effector protein is PaCpf1, LbCpf1 or AsCpf1.

14. The method of claim 1 , wherein the nucleic acid sample is obtained from a human subject.

15. The method of claim 1 , wherein the nucleic acid sample is contacted with the CRISPR-Cpf1 complex in vitro.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Zhang Feng
• Zetsche Bernd
• Gootenberg Jonathan S
• Abudayyeh Omar O
• Slaymaker Ian

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/113

  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)} · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title