US 11,920,140 B2
Organelle genome modification using polynucleotide guided endonuclease
Hajime Sakai, Newark, DE (US); Byung-Chun Yoo, Newark, DE (US); Emil Meyer Orozco, Jr., Cochranville, PA (US); Ganesh Kishore, St. Louis, MO (US); and Narendra Singh Yadav, Wilmington, DE (US)
Assigned to NAPIGEN, INC., Wilmington, DE (US)
Filed by Napigen, Inc., Wilmington, DE (US)
Filed on Jul. 18, 2022, as Appl. No. 17/867,534.
Application 17/867,534 is a division of application No. 16/109,523, filed on Aug. 22, 2018.
Claims priority of provisional application 62/548,723, filed on Aug. 22, 2017.
Prior Publication US 2023/0123175 A1, Apr. 20, 2023
Int. Cl. C12N 15/82 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01)
CPC C12N 15/8213 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/113 (2013.01); C12N 15/63 (2013.01); C12N 15/8289 (2013.01); C12N 2310/20 (2017.05); C12N 2310/3513 (2013.01); C12Y 207/07049 (2013.01)] 14 Claims
 
1. A method for altering a genome of a mitochondrion or a plastid, the method comprising:
(a) introducing into the mitochondrion or the plastid of a cell:
(i) a polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA directs a polynucleotide guided polypeptide to cleave at least one target sequence present in the genome of the mitochondrion or the plastid;
(ii) the polynucleotide guided polypeptide, wherein the polynucleotide guided polypeptide, when associated with the at least one guide RNA, cleaves the at least one target sequence; and
(iii) a replacement DNA comprising at least two regions of homology to the genome of the mitochondrion or the plastid; and
(b) selecting an altered cell comprising an altered mitochondrion genome or an altered plastid genome, wherein the altered mitochondrion genome or the altered plastid genome comprises the replacement DNA, wherein the method comprises an increase in transformation efficiency of the altered mitochondrion genome or the altered plastid genome, measured as an increase in incorporation of the replacement DNA comprising the at least two regions of homology, as compared to a control method that does not comprise the at least one guide RNA, the polynucleotide guided polypeptide, or both.