US 12,331,330 B2
Compositions and methods for RNA-templated editing in plants
Aaron Hummel, Hillsborough, NC (US); Joseph Matthew Watts, Cary, NC (US); Shai Joshua Lawit, Durham, NC (US); and Nathaniel Graham, Durham, NC (US)
Assigned to Pairwise Plants Services, Inc., Durham, NC (US)
Filed by Pairwise Plants Services, Inc., Durham, NC (US)
Filed on Oct. 23, 2020, as Appl. No. 17/078,919.
Claims priority of provisional application 62/925,097, filed on Oct. 23, 2019.
Prior Publication US 2021/0147862 A1, May 20, 2021
Int. Cl. C12N 9/22 (2006.01)
CPC C12N 9/22 (2013.01) [C07K 2319/80 (2013.01); C12N 2310/20 (2017.05)] 29 Claims
 
1. A method of modifying a target nucleic acid in a cell, the method comprising:
contacting the target nucleic acid with
a DNA binding polypeptide;
a DNA endonuclease;
a guide nucleic acid that comprises an RNA recruiting motif and a first spacer that has substantial complementarity to a first site on a first strand of the target nucleic acid, wherein the guide nucleic acid is devoid of a primer binding site, and wherein the guide nucleic acid binds to the first strand;
an extended guide nucleic acid, wherein the extended guide nucleic acid comprises (a) a second spacer having substantial complementarity to a second site on a second strand of the target nucleic acid and (b) an extended portion that comprises a primer binding site and a reverse transcriptase template encoding a modification to be incorporated into the target nucleic acid, wherein the extended guide nucleic acid binds to the second strand; and
a reverse transcriptase that binds to the RNA recruiting motif, thereby modifying the target nucleic acid in the cell,
wherein the target nucleic acid comprises the first strand and the second strand that are opposite strands of the target nucleic acid;
wherein the DNA binding polypeptide and the DNA endonuclease bind to the guide nucleic acid on the first strand;
wherein the reverse transcriptase is recruited to the second strand of the target nucleic acid by the RNA recruiting motif of the guide nucleic acid, and
wherein the reverse transcriptase polymerizes a first polynucleotide from one end of the second strand.