US 11,926,834 B2
Compositions and methods for RNA-encoded DNA-replacement of alleles
Joseph Matthew Watts, Cary, NC (US); Aaron Hummel, Hillsborough, NC (US); Yongjoo Kim, Durham, NC (US); Shai Joshua Lawit, Durham, NC (US); and David Schwark, Apex, NC (US)
Assigned to Pairwise Plants Services, Inc., Durham, NC (US)
Filed by Pairwise Plants Services, Inc., Durham, NC (US)
Filed on Nov. 5, 2020, as Appl. No. 17/090,334.
Claims priority of provisional application 62/930,836, filed on Nov. 5, 2019.
Prior Publication US 2021/0130835 A1, May 6, 2021
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/113 (2010.01)
CPC C12N 15/82 (2013.01) [C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 2310/20 (2017.05)] 15 Claims
 
1. A method of modifying a target nucleic acid, the method comprising:
contacting the target nucleic acid with
(a) a Type V CRISPR-Cas effector protein;
(b) a reverse transcriptase; and
(c) an extended guide nucleic acid, wherein the extended guide nucleic acid comprises:
(i) a Type V CRISPR nucleic acid; and
(ii) an extended portion comprising a primer binding site and a reverse transcriptase template (RT template),
wherein the extended portion of the extended guide nucleic acid comprises an RT template and a primer binding site, optionally wherein the extended portion is fused to either the 5′ end or 3′ end of the Type V CRISPR nucleic acid and/or the RT template of the extended portion is 5′ of the primer binding site, and
wherein the target nucleic acid is double stranded comprising a first strand and a second strand, the Type V CRISPR-Cas effector protein is a double stranded nuclease that cuts the first strand and the second strand of the target nucleic acid resulting in a double stranded break, and the primer binding site binds to the first strand of the target nucleic acid, which is the target strand and same strand to which the Type V CRISPR-Cas effector protein is recruited, thereby modifying the target nucleic acid.