Type V CRISPR/nuclease-system for genome editing in plant cells
AssigneeKeygene N.V.
InventorPaul Bundock
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Abstract
Provided is a method for targeted modification of DNA in a plant cell using crRNA-guided MAD7-nuclease and compositions and kits for doing the same. Preferably, the MAD7-nuclease comprises two catalytically active endonuclease domains or comprises at least one catalytically inactive endonuclease domain. Furthermore, the MAD7-endonuclease may be fused to a functional domain, such as a deaminase domain.
Claims13
A method for generating insertion and/or deletion (INDEL) mutations in DNA in a Solanum lycopersicum plant cell, comprising the step of:
- transiently introducing into the Solanum lycopersicum plant cell, a nucleic acid construct encoding a crRNA-guided MAD7-endonuclease, wherein said plant cells are protoplasts of Solanum lycopersicum,
- wherein the nucleic acid construct is introduced into the plant cell using polyethylene glycol (PEG) mediated transfection, and
- wherein efficiency of MAD7-endonuclease in generating INDEL mutations in the Solanum lycopersicum plant cell is at least 1.2 times greater than efficiency of an AsCpf1 nuclease in generating INDEL mutations in a Solanum lycopersicum plant cell.
A method for generating insertion and/or deletion (INDEL) mutations in a genome of a Solanum lycopersicum plant cell, comprising the steps of:
- transiently introducing into a population of plant cells a nucleic acid construct encoding a crRNA-guided MAD7-endonuclease;
- wherein said plant cells are protoplasts of Solanum lycopersicum and
- culturing the population of plant cells for at least 48 hours to obtain a population of plant cells wherein at least 0.5% of the population comprises the targeted modification INDEL mutations,
- wherein the nucleic acid construct is introduced into the population of plant cells using polyethylene glycol (PEG) mediated transfection, and
- wherein efficiency of MAD7-endonuclease in generating INDEL mutations in the Solanum lycopersicum plant cell is at least 1.2 times greater than efficiency of an AsCpf1 nuclease in generating INDEL mutations in a Solanum lycopersicum plant cell.
Dependencies are inferred from the claim text.