	US 12,215,329 B2
	Methods of targeted genetic alteration in plant cells
Paul Bundock, Wageningen (NL)
Assigned to Keygene N.V., Wageningen (NL)
Appl. No. 16/485,806
Filed by Keygene N.V., Wageningen (NL)
PCT Filed Feb. 15, 2018, PCT No. PCT/EP2018/053775 § 371(c)(1), (2) Date Aug. 14, 2019, PCT Pub. No. WO2018/149915, PCT Pub. Date Aug. 23, 2018.
Claims priority of application No. 2018381 (NL), filed on Feb. 15, 2017.
Prior Publication US 2020/0190527 A1, Jun. 18, 2020
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01); C12N 9/78 (2006.01); C12N 15/11 (2006.01)

CPC C12N 15/8213 (2013.01) [C12N 9/22 (2013.01); C12N 9/78 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)]

18 Claims

1. 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.