	US 11,932,859 B2
	Nuclease-mediated DNA assembly
Chris Schoenherr, Piermont, NY (US); John McWhirter, Hastings-on-Hudson, NY (US); Corey Momont, White Plains, NY (US); Caitlin L. Goshert, Boston, MA (US); Lynn Macdonald, Harrison, NY (US); Gregg S. Warshaw, Bayside, NY (US); Jose F. Rojas, Newburgh, NY (US); Ka-Man Venus Lai, Seattle, WA (US); David M. Valenzuela, Yorktown Heights, NY (US); and Andrew J. Murphy, Croton-on-Hudson, NY (US)
Assigned to Regeneron Pharmaceuticals, Inc., Tarrytown, NY (US)
Filed by REGENERON PHARMACEUTICALS, INC., Tarrytown, NY (US)
Filed on Mar. 11, 2020, as Appl. No. 16/815,194.
Application 16/815,194 is a continuation of application No. 16/296,003, filed on Mar. 7, 2019, granted, now 10,626,402.
Application 16/296,003 is a continuation of application No. 15/638,832, filed on Jun. 30, 2017, granted, now 10,273,488, issued on Apr. 3, 2019.
Application 15/638,832 is a continuation of application No. 14/747,461, filed on Jun. 23, 2015, granted, now 9,738,897, issued on Aug. 22, 2017.
Claims priority of provisional application 62/036,983, filed on Aug. 13, 2014.
Claims priority of provisional application 62/016,400, filed on Jun. 24, 2014.
Claims priority of provisional application 62/015,809, filed on Jun. 23, 2014.
Prior Publication US 2020/0208161 A1, Jul. 2, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/66 (2006.01); C12N 15/10 (2006.01); C12N 15/64 (2006.01)

CPC C12N 15/66 (2013.01) [C12N 15/1031 (2013.01); C12N 15/64 (2013.01)]

31 Claims

1. An in vitro method for assembling three or more nucleic acids, comprising:

(a) contacting a first nucleic acid with a first nuclease agent and a second nuclease agent, wherein the first nuclease agent cleaves the first nucleic acid at a first target site and the second nuclease agent cleaves the first nucleic acid at a second target site to generate a first digested nucleic acid,

wherein the first nuclease agent, the second nuclease agent, or both nuclease agents comprise a Cas9 protein and a guide RNA (gRNA) (gRNA-Cas9 complex), a zinc finger nuclease, or a Transcription Activator-Like Effector Nuclease (TALEN);

(b) contacting the first digested nucleic acid with a first joiner oligo, a second nucleic acid, a second joiner oligo, a third nucleic acid, a third joiner oligo, and an exonuclease,

wherein the first joiner oligo comprises:

(i) a first complementary sequence that is complementary to the first digested nucleic acid; and

(ii) a second complementary sequence that is complementary to the second nucleic acid; and

wherein the second joiner oligo comprises:

(i) a first complementary sequence that is complementary to the second nucleic acid; and

(ii) a second complementary sequence that is complementary to the third nucleic acid; and

wherein the third joiner oligo comprises:

(i) a first complementary sequence that is complementary to the third nucleic acid; and

(ii) a second complementary sequence that is complementary to the first digested nucleic acid; and

wherein the exonuclease exposes the complementary sequences of the first joiner oligo, the second joiner oligo, the third joiner oligo, the first digested nucleic acid, the second nucleic acid, and the third nucleic acid; and

(c) assembling the first digested nucleic acid, the first joiner oligo, the second nucleic acid, the second joiner oligo, the third nucleic acid, and the third joiner oligo.