Use of a deficient fusion protein for nuclease activity so as to induce meiotic recombinations
The present invention relates to a fusion protein comprising a domain of a nuclease from the class II CRISPR system, and a Spoll domain, wherein both domains of the fusion protein have deficient nuclease activity, as well as the use of this protein to induce targeted meiotic recombinations in a eukaryotic cell.
1 . A fusion protein, or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises:
(i) a class II type II CRISPR nuclease that has deficient nuclease activity as compared to a wild-type class II type II nuclease; and
(ii) a Spo11 protein that has deficient nuclease activity as compared to a wild-type Spo11 protein.
2 . The fusion protein of claim 1 , wherein the class II type II nuclease is a Cas9 nuclease.
3 . The fusion protein of claim 1 , wherein the class II type II nuclease comprises a polypeptide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
4 . The fusion protein of claim 3 , wherein the polypeptide sequence comprises an amino acid substitution at a position corresponding to D10 in SEQ ID NO: 38 and/or an amino acid substitution at a position corresponding to H840 in SEQ ID NO: 38.
5 . The fusion protein of claim 4 , wherein the amino acid substitution at the position corresponding to D10 in SEQ ID NO: 38 comprises an alanine substitution, and wherein the amino acid substitution at the position corresponding to H840 in SEQ ID NO: 48 comprises an alanine substitution.
6 . The fusion protein of claim 1 , wherein the class II type II CRISPR nuclease has no nuclease activity and is capable of interacting with a guide RNA.
7 . The fusion protein of claim 1 , wherein the Spo11 protein comprises a polypeptide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
8 . The fusion protein of claim 7 , wherein the polypeptide sequence comprises an amino acid substitution at a position corresponding to Y135 in SEQ ID NO: 1.
9 . The fusion protein of claim 8 , wherein the amino acid substitution at the position corresponding to Y135 in SEQ ID NO: 1 comprises a phenylalanine substitution.
10 . The fusion protein of claim 1 , wherein the Spo11 protein has no nuclease activity.
11 . The fusion protein of claim 1 , wherein the Spo11 protein is from Capsicum baccatum.
12 . A nucleic acid encoding the fusion protein of claim 1 .
13 . An expression cassette or vector comprising the nucleic acid of claim 12 operably linked to a transcriptional promoter allowing expression of the fusion protein.
14 . The expression cassette or vector of claim 13 , wherein the fusion protein is expressed during meiosis.
15 . A non-human host cell comprising the fusion protein of claim 1 or a nucleic acid encoding the fusion protein.
16 . The non-human host cell of claim 15 , wherein the host cell is a plant cell, a yeast cell or a fungal cell.
17 . A process for inducing targeted meiotic recombination(s) in a eukaryotic cell, comprising:
expressing in the eukaryotic cell:
a) the fusion protein of claim 1 ; and
b) one or more guide RNAs or one or more nucleic acids encoding the guide RNAs, wherein each of the one or more guide RNAs comprises (i) a nuclease binding RNA structure associated with a class II type II CRISPR system, and (ii) a sequence complementary to a targeted chromosomal region.
18 . The process of claim 17 , wherein the eukaryotic cell is a yeast cell.
19 . The process of claim 17 , wherein the eukaryotic cell is a plant cell.
20 . The process of claim 17 , wherein the class II type II CRISPR nuclease of the fusion protein is a Cas9 nuclease.
21 . The process of claim 17 , wherein the class II type II CRISPR nuclease of the fusion protein comprises a polypeptide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
22 . The process of claim 21 , wherein the polypeptide sequence comprises an amino acid substitution at a position corresponding to D10 in SEQ ID NO: 38 and/or an amino acid substitution at a position corresponding to H840 in SEQ ID NO: 38.
23 . The process of claim 22 , wherein the amino acid substitution at the position corresponding to D10 in SEQ ID NO: 38 comprises an alanine substitution, and wherein the amino acid substitution at the position corresponding to H840 in SEQ ID NO: 48 comprises an alanine substitution.
24 . The process of claim 17 , wherein the Spo11 protein of the fusion protein comprises a polypeptide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
25 . The process of claim 24 , wherein the polypeptide sequence comprises an amino acid substitution at a position corresponding to Y135 in SEQ ID NO: 1.
26 . The process of claim 25 , wherein the amino acid substitution at the position corresponding to Y135 in SEQ ID NO: 1 comprises a phenylalanine substitution.
27 . The process of claim 17 , wherein the Spo11 protein is from Capsicum baccatum.
28 . A process for generating variants of a eukaryotic organism, comprising:
expressing in a cell of the eukaryotic organism:
a) the fusion protein of claim 1 ; and
b) one or more guide RNAs or one or more nucleic acids encoding the guide RNAs, wherein each of the one or more guide RNAs comprises (i) a nuclease binding RNA structure associated with a class II type II CRISPR system, and (ii) a sequence complementary to a targeted chromosomal region;
obtaining cell(s) having the desired recombination(s) in the targeted chromosomal region(s); and
generating a variant of the organism from the recombinant cell(s).
29 . A process for identifying or locating genetic information encoding a trait of interest in a eukaryotic cell genome, comprising:
expressing in the eukaryotic cell:
a) the fusion protein of claim 1 ; and
b) one or more guide RNAs or one or more nucleic acids encoding the guide RNAs, wherein each of the one or more guide RNAs comprises (i) a nuclease binding RNA structure associated with a class II type II CRISPR system, and (ii) a sequence complementary to a targeted chromosomal region;
obtaining cell(s) having the desired recombination(s) in the targeted chromosomal region(s); and
analysis of the genotypes and phenotypes of the recombinant cell(s) in order to identify or locate the genetic information encoding a trait of interest.
30 . The process of claim 29 , wherein the trait of interest is a quantitative trait of interest (QTL).