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 system, 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, wherein the spacer sequence is between 25-50 nucleotides, wherein the DR sequence is an RNA sequence encoded by and corresponding to the full-length of the reverse complement sequence of SEQ ID NO: 8; and,
(2) a functional fragment of a CRISPR-associated protein (Cas), wherein said Cas has the amino acid sequence of SEQ ID NO: 1, wherein said functional fragment has an N-terminal deletion of no more than 210 residues, and a C-terminal deletion of no more than 180 residues, wherein the N-terminal deletion and C-terminal deletion result in the loss of RxxxxH motifs at both ends of the Cas having the amino acid sequence of SEQ ID NO: 1, wherein said functional fragment has completely or partially lost guide RNA-activated RNase catalytic activity compared to the guide RNA-activated RNase catalytic activity of said Cas having the amino acid sequence of SEQ ID NO: 1, and wherein said functional fragment is capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.
2 . The CRISPR-Cas system of claim 1 , wherein the target RNA is encoded by a eukaryotic DNA.
3 . The CRISPR-Cas system of claim 2 , 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.
4 . The CRISPR-Cas system of claim 1 , wherein the target RNA is an mRNA.
5 . The CRISPR-Cas system of claim 1 , wherein the spacer sequence is about 30 nucleotides.
6 . The CRISPR-Cas system of claim 1 , wherein the spacer sequence is 90-100% complementary to the target RNA.
7 . The CRISPR-Cas system of claim 1 , wherein the functional fragment has an N-terminal deletion of 180 residues, and a C-terminal deletion of 150 residues.
8 . The CRISPR-Cas system of claim 1 , wherein the functional fragment has an N-terminal deletion of about 180 residues, and a C-terminal deletion of about 150 residues.
9 . The CRISPR-Cas system of claim 8 , wherein the functional fragment is fused to an RNA base-editing domain.
10 . The CRISPR-Cas system of claim 9 , wherein the RNA base-editing domain comprises an adenosine deaminase; an APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like); or an activation-induced cytidine deaminase (AID).
11 . The CRISPR-Cas system of claim 10 , wherein the adenosine deaminase is an adenosine deaminase acting on RNA 1 (ADAR1) or a deaminase domain thereof (ADARIDD), or an adenosine deaminase acting on RNA 2 (ADAR2) or a deaminase domain thereof (ADAR2DD).
12 . The CRISPR-Cas system of claim 8 , wherein the functional fragment is fused to an RNA methyltransferase, an RNA demethylase, an RNA splicing modifier, a localization factor, or a translation modification factor.
13 . The CRISPR-Cas system of claim 8 , wherein the functional fragment is fused to a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).
14 . The CRISPR-Cas system of claim 9 , wherein the RNA base-editing domain is further fused to an RNA-binding domain.
15 . The CRISPR-Cas system of claim 11 , wherein the ADARIDD or the ADAR2DD comprises an E-to-Q mutation at a position correspondence to E488 of human ADAR2 (hADAR2).
16 . The CRISPR-Cas system of claim 11 , wherein the adenosine deaminase is a human ADAR2DD having an E488Q mutation (hADAR2DD (E488Q)).
17 . The CRISPR-Cas system of claim 1 , wherein the functional fragment is fused to an RNA methyltransferase, a RNA demethylase, an RNA splicing modifier, a localization factor, or a translation modification factor.
18 . The CRISPR-Cas system of claim 1 , wherein the functional fragment is fused to a nuclear localization signal (NLS) sequence or a nuclear export signal (NES).
19 . The CRISPR-Cas system of claim 1 , wherein the CRISPR-Cas system modifies the target RNA upon targeting the target RNA.
20 . The CRISPR-Cas system of claim 19 , wherein the modification of the target RNA is deamination of an adenosine (A) to an inosine (I).
21 . The CRISPR-Cas system of claim 1 , further comprising a target RNA comprising a sequence capable of hybridizing to the spacer sequence.
22 . The CRISPR-Cas system of claim 1 , wherein the functional fragment is fused to an RNA base-editing domain.
23 . A method of modifying a target RNA, the method comprising contacting the target RNA with the CRISPR-Cas system of claim 1 , wherein the spacer sequence is complementary to at least 15 nucleotides of the target RNA; wherein the functional fragment associates with the RNA guide sequence to form the system; wherein the system binds to the target RNA; wherein the functional fragment is fused to a heterologous functional domain; and wherein upon binding of the system to the target RNA, the functional domain modifies the target RNA.
24 . A fusion protein, comprising:
(1) a functional fragment of a CRISPR-associated protein (Cas), wherein said Cas has the amino acid sequence of SEQ ID NO: 1, wherein said 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, wherein the N-terminal deletion and C-terminal deletion result in the loss of RxxxxH motifs at both ends of the Cas having the amino acid sequence of SEQ ID NO: 1, wherein said functional fragment has completely or partially lost guide RNA-activated RNase catalytic activity compared to the guide RNA-activated RNase catalytic activity of said Cas having the amino acid sequence of SEQ ID NO: 1, and wherein said functional fragment is capable of:
(i) binding to 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
(ii) targeting the target RNA, wherein the spacer sequence is between 25-50 nucleotides, wherein the DR sequence is an RNA sequence encoded by and corresponding to the full-length of the reverse complement sequence of SEQ ID NO: 8, and
(2) a heterologous functional domain.
25 . The fusion protein of claim 24 , wherein the heterologous functional domain comprises: a nuclear localization signal (NLS), a nuclear export signal (NES), 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 HDAC, 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 an RNA ligase.
26 . An isolated eukaryotic cell comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas system, said CRISPR-Cas system 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, wherein the spacer sequence is between 25-50 nucleotides, wherein the DR sequence is an RNA sequence encoded by and corresponding to the full-length of the reverse complement sequence of SEQ ID NO: 8; and,
(2) a functional fragment of a CRISPR-associated protein (Cas), wherein said Cas has the amino acid sequence of SEQ ID NO: 1, wherein said functional fragment has an N-terminal deletion of no more than 210 residues, and a C-terminal deletion of no more than 180 residues, wherein the N-terminal deletion and C-terminal deletion result in the loss of RxxxxH motifs at both ends of the Cas having amino acid sequence of SEQ ID NO: 1, wherein said functional fragment has completely or partially lost guide RNA-activated RNase catalytic activity compared to the guide RNA-activated RNase catalytic activity of said Cas having the amino acid sequence of SEQ ID NO: 1, and wherein said functional fragment is capable of (i) binding to the RNA guide sequence and (ii) targeting the target RNA.