Induction of meiotic recombination using a crispr system

A method for inducing targeted meiotic recombination in a cell, said method comprising:

• introducing into said cell:
• a) a CRISPR nuclease or a nucleic acid encoding said nuclease, and
• b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the CRISPR nuclease and a sequence complementary to a targeted chromosomal region and
• inducing said cell to enter meiotic prophase I,
• wherein the activity of the CRISPR nuclease is repressed outside of meiosis,
• wherein the CRISPR nuclease is not fused with a Spo11 protein, and
• wherein the cell is a yeast cell, a plant cell or a fungus cell.

A method for inducing targeted meiotic recombination in a cell, said method comprising:

• introducing into said cell:
• a) a CRISPR nuclease or a nucleic acid encoding said nuclease,
• b) a plurality of guide RNAs targeting the same chromosomal region or a plurality of nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the CRISPR nuclease and a sequence complementary to a targeted chromosomal region;
• and
• c) a fusion protein comprising a Spo11 protein operably linked to a DNA binding domain, or a nucleic acid encoding said fusion protein, optionally under the control of a meiosis-specific promoter;
• and
• inducing said cell to enter meiotic prophase I,
• wherein the CRISPR nuclease is not fused with a Spo11 protein,
• optionally wherein the activity of the CRISPR nuclease is repressed outside of meiosis, and
• wherein the cell is a yeast cell, a plant cell or a fungus cell.

A method for generating variants of an organism, said method comprising:

• introducing into a cell of said organism:
• a) a CRISPR nuclease or a nucleic acid encoding said nuclease, and
• b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the CRISPR nuclease and a sequence complementary to a targeted chromosomal region and
• inducing said cell to enter meiotic prophase I,
• obtaining a cell or cells having the desired recombination(s) at the targeted chromosomal region(s);
• and
• generating a variant of the organism from said recombinant cell,
• wherein the activity of the CRISPR nuclease is repressed outside of meiosis,
• wherein the CRISPR nuclease is not fused with a Spo11 protein, and
• wherein the organism is a yeast, a plant or a fungus.

A method for generating variants of an organism, said method comprising:

• introducing into a cell of said organism:
• a) a CRISPR nuclease or a nucleic acid encoding said nuclease, and
• b) a plurality of guide RNAs targeting the same chromosomal region or a plurality of nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the CRISPR nuclease and a sequence complementary to a targeted chromosomal region and
• inducing said cell to enter meiotic prophase I,
• obtaining a cell or cells having the desired recombination(s) at the targeted chromosomal region(s);
• and
• generating a variant of the organism from said recombinant cell,
• wherein the CRISPR nuclease is not fused with a Spo11 protein and wherein the activity of the CRISPR nuclease is repressed outside of meiosis, and
• wherein the organism is a yeast, a plant or a fungus.

A host cell comprising:

• a) a CRISPR nuclease or a nucleic acid encoding said nuclease, and
• b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the CRISPR nuclease and a sequence complementary to a targeted chromosomal region, and
• c) optionally a fusion protein comprising a Spo11 protein operably linked to a DNA binding domain, or a nucleic acid encoding said fusion protein,
• wherein the CRISPR nuclease is not fused with a Spo11 protein and
• wherein the activity of the CRISPR nuclease is repressed outside of meiosis by
• (i) placing the expression of said CRISPR nuclease and/or said guide RNAs under the control of a promoter inducible during the meiotic phase or a meiosis-specific promoter, and/or
• (ii) expressing an inhibitor of CRISPR endonuclease activity outside of meiosis, and/or
• (iii) using a system triggering the degradation of the CRISPR nuclease outside of meiosis, and/or
• (iv) using a CRISPR nuclease which is a variant of a wild-type CRISPR nuclease, said variant exhibiting a reduced endonuclease activity by comparison to said wild-type CRISPR nuclease;
• wherein the host cell is a yeast cell, a plant cell or a fungus cell.