Granted Patent
B2
US 12,275,952 · App. 16/277,638 · Granted Apr 15, 2025
Using programmable DNA binding proteins to enhance targeted genome modification
Assignee:
Sigma-Aldrich Co. LLC
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Abstract
Compositions and methods for using programmable DNA binding proteins to increase the efficiency and/or specificity of targeted genome modification or to facilitate the detection of specific genomic loci in eukaryotic cells.
Claims (16)
1. A composition for introduction into a eukaryotic cell, the composition comprising:
(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;
(b) at least one CRISPR system having binding activity but lacking cleavage activity, or a nucleic acid encoding said at least one 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;
wherein the CRISPR system of subpart (a) is capable of being targeted to a target chromosomal sequence within a target chromosomal region and the at least one CRISPR system of subpart (b) is capable of being targeted to and, excluding any off-target chromosomal sequence within the target chromosomal region, such that the CRISPR systems of subparts (a) and (b) are capable of being 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 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 is capable of being exchanged with or integrated into the chromosomal sequence at the target chromosomal sequence.
2. The composition of claim 1 , wherein the CRISPR systems of subparts (a) and (b) are capable of being located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 100 base pairs of one another on the target chromosomal region.
3. The composition of claim 1 , wherein the CRISPR systems of subparts (a) and (b) are capable of being located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 75 base pairs of one another on the target chromosomal region.
4. The composition of claim 1 , wherein the CRISPR systems of subparts (a) and (b) are capable of being located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 50 base pairs of one another on the target chromosomal region.
5. The composition of claim 1 , wherein the CRISPR systems of subparts (a) and (b) are capable of being located in spatial proximity only by the chromosomal DNA binding action of their respective guide RNA and are within about 25 base pairs of one another on the target chromosomal region.
6. The composition of claim 1 , wherein nucleic acid encoding each CRISPR system of subpart (a) and subpart (b) is mRNA or DNA.
7. The composition of claim 1 , wherein nucleic acid encoding each CRISPR system of subpart (a) and subpart (b) and/or encoding each guide RNA of subpart (a) and subpart (b) is part of a plasmid vector or a viral vector.
8. The composition of claim 1 , wherein the CRISPR system of subpart (a) comprises a catalytically active Type V CRISPR/Cpf1 protein and the at least one CRISPR system of subpart (b) comprises a catalytically inactive Type II CRISPR/Cas9 protein.
9. The composition of claim 1 , wherein the CRISPR system of subpart (a) comprises a catalytically active Type II CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) comprises a catalytically inactive Type II CRISPR/Cas9 protein.
10. The composition of claim 1 , comprising two CRISPR systems having binding activity but lacking cleavage activity.
11. The composition of claim 1 , wherein (i) the CRISPR system of subpart (a) is a Type IIB Francisella novicida CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIA Streptococcus pyogenes CRISPR/Cas9 protein; (ii) the CRISPR system of subpart (a) is a Type IIA Streptococcus pyogenes CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIB Francisella novicida CRISPR/Cas9 protein; (iii) the CRISPR system of subpart (a) is a Type IIC Campylobacter jejuni CRISPR/Cas9 protein and the at least one CRISPR system of subpart (b) is a Type IIA Streptococcus pyogenes CRISPR/Cas9 protein; or (iv) the CRISPR system of subpart (a) is a Type V Francisella novicida CRISPR/Cpf1 protein and the at least one CRISPR system of subpart (b) is a Type IIA Streptococcus pyogenes CRISPR/Cas9 protein.
12. A kit comprising the composition of claim 1 and one or more components selected from transfection reagents, cell growth media, selection media, in-vitro transcription reagents, nucleic acid purification reagents, protein purification reagents, buffers, and instructions for use.
Continuity (4)
Provisional Application
62358415
· Jul 5, 2016
Provisional Application
62344858
· Jun 2, 2016
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