US 12,084,675 B2
Using programmable DNA binding proteins to enhance targeted genome modification
Fuqiang Chen, St. Louis, MO (US)
Assigned to Sigma-Aldrich Co. LLC, St. Louis, MO (US)
Filed by SIGMA-ALDRICH CO. LLC, St. Louis, MO (US)
Filed on Feb. 15, 2019, as Appl. No. 16/277,614.
Application 16/277,614 is a continuation of application No. 15/437,148, filed on Feb. 20, 2017, granted, now 10,266,851.
Claims priority of provisional application 62/358,415, filed on Jul. 5, 2016.
Claims priority of provisional application 62/344,858, filed on Jun. 2, 2016.
Prior Publication US 2019/0169650 A1, Jun. 6, 2019
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01)
CPC C12N 15/907 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01)] 11 Claims
 
1. A method for increasing targeted double-stranded cleavage activity and/or specificity at a target chromosomal region in a eukaryotic cell, the method comprising introducing into the eukaryotic cell:
(a) a CRISPR system having binding activity and double-stranded cleavage activity, or a nucleic acid encoding said CRISPR system, comprising (i) a catalytically active Type II CRISPR/Cas9 protein or a catalytically active Type V CRISPR/Cpf1 protein, and (ii) a guide RNA; and
(b) at least one CRISPR system having binding activity but lacking cleavage activity, or a nucleic acid encoding said CRISPR system, comprising (i) a catalytically inactive Type II CRISPR/Cas9 protein or a catalytically inactive Type V CRISPR/Cpf1 protein, and (ii) a guide RNA; and
(c) a donor polynucleotide comprising a donor sequence, wherein the donor sequence is for targeted integration of a sequence and is introduced into said eukaryotic cell separately from the (a)(ii) guide RNA and the b(ii) guide RNA;
wherein the CRISPR system of subpart (a) is targeted to a target chromosomal sequence within the target chromosomal region and the at least one CRISPR system of subpart (b) is targeted to and, excluding any off-target chromosomal binding, binds solely to a site that is proximal to the target chromosomal sequence within the target chromosomal region, such that the CRISPR systems of subparts (a) and (b) are located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 200 base pairs of one another on the target chromosomal region, and
wherein binding of the at least one CRISPR system of subpart (b) to the site proximal to the target chromosomal sequence increases accessibility of the CRISPR system of subpart (a) to the target chromosomal sequence, thereby increasing targeted double-stranded cleavage activity and/or specificity;
and wherein the donor sequence is flanked by sequences having substantial sequence identity to sequences located on either side of the target chromosomal sequence, such that during repair of the double-stranded break by a homology directed repair process (HDR) the donor sequence in the donor polynucleotide can be exchanged with or integrated into the chromosomal sequence at the target chromosomal sequence.