Novel crispr enzymes and systems

Patent metadata

Field	Value
Publication number	US-2016208243-A1
Application number	US-201514975085-A
Country	US
Kind code	A1
Filing date	Dec 18, 2015
Priority date	Jun 18, 2015
Publication date	Jul 21, 2016
Grant date	—

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.

First claim

What is claimed:

1 . An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
e) one or more Type V CRISPR-Cas polynucleotide sequences comprising a guide RNA which comprises a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more Type V CRISPR-Cas polynucleotide sequences, and f) a Cpf1 effector protein, or one or more nucleotide sequences encoding the Cpf1 effector protein; wherein the one or more guide sequences hybridize to said target sequence, said target sequence is 3′ of a Protospacer Adjacent Motif (PAM), and said guide RNA forms a complex with the Cpf1 effector protein, wherein the system comprises Mg 2+ .

2 . An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising
g) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more Type V CRISPR-Cas polynucleotide sequences comprising a guide RNA which comprises a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, h) a second regulatory element operably linked to a nucleotide sequence encoding a Cpf1 effector protein; wherein components (a) and (b) are located on the same or different vectors of the system, wherein when transcribed, the one or more guide sequences hybridize to said target sequence, said target sequence is 3′ of a Protospacer Adjacent Motif (PAM), and said guide RNA forms a complex with the Cpf1 effector protein, wherein the system comprises Mg 2+ .

3 . The system of claim 1 or 2 wherein the target sequences is within a cell.

4 . The system of claim 3 wherein the cell comprises a eukaryotic cell.

5 . The system according to claim 1 or 2 , wherein when transcribed the one or more guide sequences hybridize to the target sequence and the guide RNA forms a complex with the Cpf1 effector protein which causes cleavage distally of the target sequence.

6 . The system according to claim 5 , wherein said cleavage generates a staggered double stranded break with a 4 or 5-nt 5′ overhang.

7 . The system according to claim 1 or 2 , wherein the PAM comprises a 5′ T-rich motif.

8 . The system according to claim 1 or 2 , wherein the effector protein is a Cpf1 effector protein derived from a bacterial species listed in FIG. 64 .

9 . The system according to claim 8 , wherein the Cpf1 effector protein is derived from 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.

10 . The system according to claim 9 , 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 PaCpf1p, LbCpf1 or AsCpf1.

11 . The system according to claim 1 or 2 , wherein the Cpf1 effector protein comprises one or more nuclear localization signals.

12 . The system according to claim 1 or 2 , wherein the nucleic acid sequences encoding the Cpf1 effector protein is codon optimized for expression in a eukaryotic cell.

13 . The system according to claim 1 or 2 wherein components (a) and (b) or the nucleotide sequences are on one vector.

14 . A method of modifying a target locus of interest comprising delivering a system according to claim 1 or 2 , to said locus or a cell containing the locus.

15 . A method of modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Cpf1 effector protein and one or more nucleic acid components, wherein the Cpf1 effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to a target locus of interest that is 3′ of a Protospacer Adjacent Motif (PAM), the effector protein induces a modification of the target locus of interest, wherein the complex comprises Mg 2+ .

16 . The method of claim 15 , wherein the target locus of interest is within a cell.

17 . The method of claim 16 , wherein the cell is a eukaryotic cell.

18 . The method of claim 16 , wherein the cell is an animal or human cell.

19 . The method of claim 16 , wherein the cell is a plant cell.

20 . The method of claim 15 , wherein the target locus of interest is comprised in a DNA molecule in vitro.

21 . The method of claim 15 , wherein said non-naturally occurring or engineered composition comprising a Cpf1 effector protein and one or more nucleic acid components is delivered to the cell as one or more polynucleotide molecules.

22 . The method of claim 15 , wherein the target locus of interest comprises DNA.

23 . The method of claim 22 , wherein the DNA is relaxed or supercoiled.

24 . The method of claim 15 , wherein the composition comprises a single nucleic acid component.

25 . The method of claim 24 , wherein the single nucleic acid component comprises a guide sequence linked to a direct repeat sequence.

26 . The method of claim 15 wherein the modification of the target locus of interest is a strand break.

27 . The method of claim 26 , wherein the strand break comprises a staggered DNA double stranded break with a 4 or 5-nt 5′ overhang.

28 . The method of claim 26 , wherein the target locus of interest is modified by the integration of a DNA insert into the staggered DNA double stranded break.

29 . The method of claim 15 , wherein the Cpf1 effector protein comprises one or more nuclear localization signal(s) (NLS(s)).

30 . The method of claim 21 , wherein the one or more polynucleotide molecules are comprised within one or more vectors.

31 . The method of claim 21 , wherein the one or more polynucleotide molecules comprise one or more regulatory elements operably configured to express the Cpf1 effector protein and/or the nucleic acid component(s), optionally wherein the one or more regulatory elements comprise inducible promoters.

32 . The method of claim 21 wherein the one or more polynucleotide molecules or the one or more vectors are comprised in a delivery system.

33 . The method of claim 21 , wherein system or the one or more polynucleotide molecules are delivered via particles, vesicles, or one or more viral vectors.

34 . The method of claim 33 wherein the particles comprise a lipid, a sugar, a metal or a protein.

35 . The method of claim 33 wherein the vesicles comprise exosomes or liposomes.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

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

Classifications

• C12N15/8201

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title

• C12N2310/3519

  Fusion with another nucleic acid · CPC title

• C12N2310/16

  Aptamers · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N9/22 Primary

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