Type VI-E and type VI-F CRISPR-Cas system and uses thereof
The invention provides novel CRISPR/Cas compositions and uses thereof for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting Cas13e or Cas13f effector protein, and at least one targeting nucleic acid component such as a guide RNA (gRNA) or crRNA. The novel Cas effector proteins are among the smallest of the known Cas effector proteins, at about 800 amino acids in size, and are thus uniquely suitable for delivery using vectors of small capacity, such as an AAV vector.
1. A Clustered Regularly interspaced Short Palindromic Repeat (CRISPR)-Cas complex, comprising:
(1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA and a direct repeat (DR) sequence 3′ to the spacer sequence; and,
(2) a CRISPR-associated protein (Cas) having;
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) an amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) a functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7, wherein the functional fragment consists of an N-terminal and/or a C-terminal deletion and is at least 50% Identical to any one of SEQ ID Nos: 2-7,
wherein
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; and
c) the functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7,
are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA,
with the proviso that the spacer sequence is not 100% complementary to a naturally-occurring bacteriophage nucleic acid when the complex comprises the Cas amino acid sequence of any one of SEQ ID Nos: 2-7.
2. The CRISPR-Cas complex of claim 1 , wherein the DR sequence has substantially the same secondary structure as the secondary structure of a DR sequence encoded by a complement sequence of any one of SEQ ID NOs: 9-14.
3. The CRISPR-Cas complex of claim 1 , wherein the DR sequence is encoded by a complement sequence of any one of SEQ ID NOs: 9-14.
4. The CRISPR-Cas complex of claim 1 , wherein the target RNA is encoded by a eukaryotic DNA.
5. The CRISPR-Cas complex of claim 4 , wherein the eukaryotic DNA is a non-human mammalian DNA, a non-human primate DNA, a human DNA, a plant DNA, an insect DNA, a bird DNA, a reptile DNA, a rodent DNA, a fish DNA, a worm/nematode DNA, or a yeast DNA.
6. The CRISPR-Cas complex of claim 1 , wherein the target RNA is an mRNA.
7. The CRISPR-Cas complex of claim 1 , wherein the spacer sequence is between 15-60 nucleotides, between 25-50 nucleotides, or about 30 nucleotides.
8. The CRISPR-Cas complex of claim 1 , wherein the spacer sequence is 90-100% complementary to the target RNA.
9. The CRISPR-Cas complex of claim 1 , wherein the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 comprises conserved amino acid substitutions of one or more residues of any one of SEQ ID NOs. 2-7.
10. The CRISPR-Cas complex of claim 9 , wherein the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 comprises only conserved amino acid substitutions.
11. The CRISPR-Cas complex of claim 1 , wherein the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 has identical sequence to wild-type Cas of any one of SEQ ID NOs: 2-7 in an HEPN domain or an RXXXXH motif.
12. The CRISPR-Cas complex of claim 1 , wherein the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 is capable of binding to the RNA guide sequence when said RNA guide sequence is hybridized to the target RNA, and wherein said amino acid sequence at least about 90°/o identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 has no RNase catalytic activity due to a mutation in the RNase catalytic site of the Cas.
13. The CRISPR-Cas complex of claim 1 , wherein the functional fragment has an N-terminal deletion of no more than 210 residues, and/or a C-terminal deletion of no more than 180 residues.
14. The CRISPR-Cas complex of claim 13 , wherein the functional fragment has an N-terminal deletion of about 180 residues, and/or a C-terminal deletion of about 150 residues.
15. The CRISPR-Cas complex of claim 14 , wherein the functional fragment is fused to an RNA base-editing domain.
16. The CRISPR-Cas complex of claim 15 , wherein the RNA base-editing domain comprises an adenosine deaminase; an apolipoprotein B mRNA editing enzyme; a catalytic polypeptide-like (APOBEC); or an activation-induced cytidine deaminase (AID).
17. The CRISPR-Cas complex of claim 16 , wherein the adenosine deaminase is adenosine deaminase acting on RNA 2 (ADAR2) and wherein the ADAR2 has an E488Q/T375G double mutation or is ADAR2DD.
18. The CRISPR-Cas complex of claim 15 , wherein the base-editing domain is further fused to an RNA-binding domain.
19. The CRISPR-Cas complex of claim 12 , wherein the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7 is fused to an RNA methyltransferase, a RNA demethylase, an RNA splicing modifier, a localization factor, or a translation modification factor.
20. The CRISPR-Cas complex of claim 1 , wherein
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) the functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7
is fused to a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).
21. The CRISPR-Cas complex of claim 1 , wherein targeting of the target RNA results in a modification of the target RNA.
22. The CRISPR-Cas complex of claim 21 , wherein the modification of the target RNA is a cleavage of the target RNA.
23. The CRISPR-Cas complex of claim 21 , wherein the modification of the target RNA is deamination of an adenosine (A) to an inosine (I).
24. The CRISPR-Cas complex of claim 1 , further comprising a target RNA comprising a sequence capable of hybridizing to the spacer sequence.
25. A fusion protein, comprising
a CRISPR-associated protein (Cas) having:
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) an amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) afunctional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7, wherein the functional fragment consists of an N-terminal and/or a C-terminal deletion and is at least 50% identical to any one of SEQ ID Nos: 2-7, wherein the functional fragment is capable of (i) binding to an RNA guide sequence and (ii) targeting a target RNA,
and
(2) a heterologous functional domain.
26. The fusion protein of claim 25 , wherein the heterologous functional domain comprises: a nuclear localization signal (NLS), a reporter protein, a detection label, a localization signal, a protein targeting moiety, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription inhibition domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HBAC, a polypeptide having ssRNA cleavage activity, a polypeptide having dsRNA cleavage activity, a polypeptide having ssDNA cleavage activity, a polypeptide having dsDNA cleavage activity, a DNA ligase, or gi RNA ligase, or any combination thereof.
27. A method of modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas complex of claim 1 , wherein the spacer sequence is complementary to at least 15 nucleotides of the target RNA; wherein
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) the functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7
associates with the RNA guide sequence to form the complex; wherein the complex binds to the target RNA; and wherein upon binding of the complex to the target RNA,
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) the functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7
modifies the target RNA.
28. The method of claim 27 , wherein the target RNA is modified by deamination by a Double-stranded RNA-specific adenosine deaminase fused to the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7.
29. The method of claim 27 , wherein the target RNA is an mRNA, a tRNA, an rRNA, a non-coding RNA, an lncRNA, or a nuclear RNA.
30. An isolated eukaryotic cell comprising a Clustered Regularly interspaced Short Palindromic Repeat (CRISPR)-Cas complex, comprising:
(1) an RNA guide sequence comprising a spacer sequence capable of hybridizing to a target RNA and a direct repeat (DR) sequence 3′ to the spacer sequence; and,
a CRISPR-associated protein (Cas) having:
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) an amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; or
c) a functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7, wherein the functional fragment consists of an N-terminal and/or a C-terminal deletion and is at least 50% identical to any one of SEQ ID Nos: 2-7,
wherein
a) the amino acid sequence of any one of SEQ ID Nos: 2-7;
b) the amino acid sequence at least about 90% identical to the amino acid sequence of any one of SEQ ID Nos: 2-7; and
c) the functional fragment of the amino acid sequence of any one of SEQ ID Nos: 2-7,
are capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.