Compositions and methods for RNA-encoded DNA-replacement of alleles
This invention relates to recombinant nucleic constructs comprising Type II CRISPR-Cas effector proteins, reverse transcriptases and extended guide nucleic acids and methods of use thereof for modifying nucleic acids in plants.
1 . A method of modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with
(a) a Type II CRISPR-Cas effector protein;
(b) a reverse transcriptase; and
(c) an extended guide nucleic acid, wherein the extended guide nucleic acid comprises:
(i) a Type II CRISPR nucleic acid; and
(ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template),
wherein the extended portion of the extended guide nucleic acid comprises an RT template and a primer binding site, optionally wherein the extended portion is fused to either the 5′ end or 3′ end of the Type II CRISPR nucleic acid and/or the RT template of the extended portion is 5′ of the primer binding site,
wherein the target nucleic acid is double stranded comprising a first strand and a second strand, the Type II CRISPR-Cas effector protein is a double stranded nuclease that cuts the first strand and the second strand of the target nucleic acid resulting in a double stranded break, and the primer binding site binds to the first strand of the target nucleic acid, which is the target strand and same strand to which the Type II CRISPR-Cas effector protein is recruited, thereby modifying the target nucleic acid,
wherein the primer binding site has length of about eight nucleotides to about 100 nucleotides, and
wherein the RT template has a length of about seven to about 100 nucleotides.
2 . The method of claim 1 , wherein the primer binding site is at least 45 nucleotides in length, or about 45 nucleotides to about 100 nucleotides.
3 . The method of claim 1 , wherein the RT template is a length of about seven nucleotides to about 40 nucleotides.
4 . The method of claim 1 , wherein the extended portion of the extended guide RNA is linked to the Type II CRISPR nucleic acid and/or the tracrRNA via a linker, optionally wherein the linker is 1 to 100 nucleotides in length.
5 . The method of claim 1 , wherein when the extended portion is located 5′ of the crRNA, the Type II CRISPR-Cas effector protein is modified to reduce or eliminate self-processing RNAse activity.
6 . The method of claim 1 , further comprising contacting the target nucleic acid with a Dna2 polypeptide and/or a 5′ flap endonuclease (FEN), optionally wherein the FEN and/or Dna2 polypeptide is overexpressed (in the presence of the target nucleic acid), and/or optionally wherein the FEN is a fusion protein comprising an FEN domain fused to the Type II CRISPR-Cas effector protein and/or wherein the Dna2 polypeptide is a fusion protein comprising an Dna2 domain fused to the or Type II CRISPR-Cas effector protein.
7 . A method of modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with
(a) a Type II CRISPR-Cas effector protein;
(b) a reverse transcriptase, and
(c) an extended guide nucleic acid, wherein the extended guide nucleic acid comprises:
(i) a Type II CRISPR nucleic acid; and
(ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template),
wherein the extended portion of the extended guide nucleic acid comprises an RT template and a primer binding site, optionally wherein the extended portion is fused to either the 5′ end or 3′ end of the Type II CRISPR nucleic acid and/or the RT template of the extended portion is 5′ of the primer binding site,
wherein the target nucleic acid is double stranded comprising a first strand and a second strand, the Type II CRISPR-Cas effector protein is a double stranded nuclease that cuts the first strand and the second strand of the target nucleic acid resulting in a double stranded break, and the primer binding site binds to the first strand of the target nucleic acid, which is the target strand and same strand to which the Type II CRISPR-Cas effector protein is recruited,
wherein the Type II CRISPR-Cas effector protein is a Type II CRISPR-Cas effector fusion protein and/or the reverse transcriptase is a reverse transcriptase fusion protein, wherein the Type II CRISPR-Cas fusion protein, the reverse transcriptase fusion protein and/or the extended guide nucleic acid is fused to one or more components that recruit the reverse transcriptase to the Type II CRISPR-Cas effector protein, optionally the one or more components recruit via protein-protein interactions, protein-RNA interactions, and/or chemical interactions, thereby modifying the target nucleic acid,
wherein the primer binding site has length of about eight nucleotides to about 100 nucleotides, and
wherein the RT template has a length of about seven to about 100 nucleotides.
8 . The method of claim 7 , wherein the Type II CRISPR-Cas effector protein is a Type II CRISPR-Cas effector fusion protein comprising a Type II CRISPR-Cas effector protein domain fused to a peptide tag and the reverse transcriptase is a reverse transcriptase fusion protein comprising a reverse transcriptase domain fused (linked) to an affinity polypeptide that binds to the peptide tag, optionally wherein the target nucleic acid is contacted with two or more reverse transcriptase fusion proteins, optionally wherein the peptide tag comprises a GCN4 peptide tag, a c-Myc affinity tag, an HA affinity tag, a His affinity tag, an S affinity tag, a methionine-His affinity tag, an RGD-His affinity tag, a FLAG octapeptide, a strep tag or strep tag II, a V5 tag, and/or a VSV-G epitope and/or the affinity polypeptide is an antibody, an affibody, an anticalin, a monobody and/or a DARPin, optionally wherein the antibody is an scFv antibody.
9 . The method of claim 8 , wherein the peptide tag comprises 2 or more copies of the peptide tag.
10 . The method of claim 8 , wherein the extended guide nucleic acid is linked to an RNA recruiting motif, and the reverse transcriptase is a reverse transcriptase fusion protein comprising a reverse transcriptase domain fused to an affinity polypeptide that binds to the RNA recruiting motif, optionally wherein the target nucleic acid is contacted with two or more reverse transcriptase fusion proteins, optionally wherein the extended guide RNA is linked to two or more RNA recruiting motifs, optionally wherein the RNA recruiting motif and corresponding affinity polypeptide are a telomerase Ku binding motif and the affinity polypeptide of Ku; a telomerase Sm7 binding motif and the affinity polypeptide of Sm7; an MS2 phage operator stem-loop and the affinity polypeptide MS2 Coat Protein (MCP), a PP7 phage operator stem-loop and the affinity polypeptide PP7 Coat Protein (PCP); an SfMu phage Com stem-loop and the affinity polypeptide Com RNA binding protein; a PUF binding site (PBS) and the affinity polypeptide Pumilio/fem-3 mRNA binding factor (PUF); and/or a synthetic RNA-aptamer and the corresponding aptamer ligand.
11 . A method of modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with
(a) a Type II CRISPR-Cas effector protein;
(b) a reverse transcriptase;
(c) an extended guide nucleic acid, wherein the extended guide nucleic acid comprises:
(i) a Type II CRISPR nucleic acid; and
(ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template); and
(d) a 5′-3′ exonuclease,
wherein the extended portion of the extended guide nucleic acid comprises an RT template and a primer binding site, optionally wherein the extended portion is fused to either the 5′ end or 3′ end of the Type II CRISPR nucleic acid and/or the RT template of the extended portion is 5′ of the primer binding site,
wherein the target nucleic acid is double stranded comprising a first strand and a second strand, the Type II CRISPR-Cas effector protein is a double stranded nuclease that cuts the first strand and the second strand of the target nucleic acid resulting in a double stranded break, and the primer binding site binds to the first strand of the target nucleic acid, which is the target strand and same strand of the target nucleic acid to which the Type II CRISPR-Cas effector protein is recruited, thereby modifying the target nucleic acid,
wherein the primer binding site has length of about eight nucleotides to about 100 nucleotides, and
wherein the RT template has a length of about seven to about 100 nucleotides.
12 . The method of claim 11 , wherein the 5′-3′ exonuclease is fused to a Type II CRISPR-Cas effector protein, optionally wherein the Type II CRISPR-Cas effector protein is a Type II CRISPR-Cas fusion protein.
13 . The method of claim 11 , wherein (a) the 5′-3′ exonuclease is a fusion protein comprising the 5′-3′ exonuclease fused to a peptide tag and the Type II CRISPR-Cas effector protein is a fusion protein comprising a Type II CRISPR-Cas effector protein domain fused to an affinity polypeptide that is capable of binding to the peptide tag, (b) the 5′-3′ exonuclease is a fusion protein comprising the 5′-3′ exonuclease fused to an affinity polypeptide that is capable of binding to the peptide tag and the Type II CRISPR-Cas effector protein is a fusion protein comprising a Type II CRISPR-Cas effector protein domain fused to a peptide tag, or (c) the 5′-3′ exonuclease is a fusion protein comprising the 5′-3′ exonuclease fused to an affinity polypeptide that is capable of binding to an RNA recruiting motif and the extended guide nucleic acid is linked to an RNA recruiting motif.
14 . The method of claim 11 , wherein the 5′-3′ exonuclease is a RecE exonuclease, a RecJ exonuclease, a T5 exonuclease or a T7exonuclease.
15 . The method of claim 11 , further comprising contacting the target nucleic acid with a Dna2 polypeptide and/or a 5′ flap endonuclease (FEN), optionally wherein the FEN and/or Dna2 polypeptide is overexpressed in the presence of the target nucleic acid, and/or optionally wherein the FEN is a fusion protein comprising an FEN domain fused to the Type II CRISPR-Cas effector protein and/or wherein the Dna2 polypeptide is a fusion protein comprising an Dna2 domain fused to the Type II CRISPR-Cas effector protein.