Patent ID: 11932859
Assignee: REGENERON PHARMACEUTICALS, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.