Nuclease-mediated DNA assembly

摘要

Methods are provided herein for assembling at least two nucleic acids using a sequence specific nuclease agent (e.g., a gRNA-Cas complex) to create end sequences having complementarity and subsequently assembling the overlapping complementary sequences. The nuclease agent (e.g., a gRNA-Cas complex) can create double strand breaks in dsDNA in order to create overlapping end sequences or can create nicks on each strand to produce complementary overhanging end sequences. Assembly using the method described herein can assemble any nucleic acids having overlapping sequences or can use a joiner oligo to assemble sequences without complementary ends.

主权项

1. An in vitro method for seamlessly assembling two or more double-stranded nucleic acids, comprising:
(a) contacting a first nucleic acid with at least one nuclease agent, wherein the at least one nuclease agent cleaves the first nucleic acid at a first target site to generate a first digested nucleic acid, wherein the cleaving removes a double-stranded fragment from the end of the first nucleic acid at which the seamless assembly will occur; (b) contacting the first digested nucleic acid with a second nucleic acid, a double stranded joiner oligo, and an exonuclease, wherein the joiner oligo is a linear double-stranded DNA that is from about 50 bp to about 400 bp and comprises: (i) a first complementary sequence that is complementary to the first digested nucleic acid; (ii) a spacer; and (iii) a second complementary sequence that is complementary to the second nucleic acid, wherein the spacer comprises a sequence identical to the fragment, wherein no nucleic acid bases are present between the first complementary sequence and the sequence identical to the fragment, and no nucleic acid bases are present between the second complementary sequence and the sequence identical to the fragment; wherein the exonuclease exposes the first and second complementary sequences; and (c) assembling the joiner oligo with the first digested nucleic acid and the second nucleic acid, wherein the assembly reconstitutes the fragment.