Methods of targeted genetic alteration in plant cells
AssigneeKeygene N.V.
InventorPaul Bundock
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Abstract
The current invention relates to methods of targeted genetic alteration in cells, preferably plant cells, as well as to plant cells and plants thus obtained using at least a fusion protein comprising a site-specific nuclease domain and a deaminase domain, or a construct encoding the same. The method also provides for a composition and a kit comprising a combination of a first fusion protein comprising a cytosine deaminase domain and a second fusion protein comprising an adenine deaminase domain, preferably for use in the method of the invention. The method provides for targeted alteration of a DNA duplex in plant cells with increased efficacy.
Claims18
A method for targeted conversion of one or more cytosines to thymines (C-to-T) in combination with one or more adenines to guanines (A-to-G) in a target sequence in a cell, comprising
- i) contacting DNA in the cell with at least a first and a second fusion protein,
- wherein the first fusion protein comprises a CRISPR-nuclease domain and a cytosine deaminase domain and wherein the second fusion protein comprises a CRISPR-nuclease domain and an adenine deaminase domain,
- wherein the DNA is contacted transiently by at least one of:
- transiently introducing into the cell one or more DNA constructs for the combined expression of the first and second fusion proteins;
- and
- transiently introducing into the cell the first and second fusion proteins,
- wherein the cell is transfected using a transfection medium comprising a guide RNA and both the first and the second fusion protein, or construct(s) encoding the same,
- wherein the first and second fusion protein comprise the same CRISPR-nuclease domain;
- and
- ii) sequencing the DNA to identify the C-to-T and A-to-G conversions in the target sequence.
Dependencies are inferred from the claim text.