received by the International Bureau on 11 January 2016 (11.01.16)

1. A method for assembling at least two nucleic acids, comprising:

(a) contacting a first nucleic acid with a first nuclease agent, wherein the first nuclease agent cleaves the first nucleic acid at a first target site to produce a first digested nucleic acid with overlapping end sequences between the first digested nucleic acid and a second nucleic acid;

(b) contacting the first digested nucleic acid and the second nucleic acid with an exonuclease to expose complementary sequences between the first digested nucleic acid and the second nucleic acid; and

(c) assembling the two nucleic acid fragments generated from step (b).

2. The method of claim 1, wherein step (c) further comprises:

(i) annealing the exposed complementary sequences;

(ii) extending the 3' ends of the annealed complementary sequences; and

(iii) ligating the first and the second nucleic acids.

3. The method of claim 1 or 2, wherein step (a) further comprises contacting the second nucleic acid with a second nuclease agent, wherein the second nucleic acid does not comprise the overlapping end sequence, and the second nuclease agent cleaves the second nucleic acid at a second target site to produce a second digested nucleic acid with the overlapping end sequences between the first digested nucleic acid and the second digested nucleic acid, and

wherein the second nucleic acid of step (b) is the second digested nucleic acid.

4. The method of claim 3, wherein at least one of the first nuclease agent and the second nuclease agent comprises a Cas protein and a guide RNA (gRNA) (gRNA-Cas complex), a zinc finger nuclease, or a Transcription Activator-Like Effector Nuclease (TALEN) that targets the first target site or the second target site.

5. The method of claim 4, wherein at least one of the first nuclease agent and the second nuclease agent comprises the Cas protein and the guide RNA (gRNA) (gRNA-Cas complex), wherein the Cas protein is a Cas9 protein, the gRNA comprises a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), and at least one of the first target site and the second target site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

6. The method of claim 5, wherein the Cas9 protein comprises a RuvC domain and a HNH domain, at least one of which lacks endonuclease activity.

7. The method of any one of claims 1-6, wherein the overlapping end sequence ranges from 20 bp to 200 bp long.

8. The method of any one of claims 1-7, wherein the first nucleic acid, the second nucleic acid, or both nucleic acids are derived from a bacterial artificial chromosome.

9. The method of claim 8, wherein the bacterial artificial chromosome comprises a human DNA, a rodent DNA, a synthetic DNA, a human polynucleotide sequence, or a combination thereof.

10. A method for assembling at least two nucleic acids, comprising:

(a) contacting a first nucleic acid with a first nuclease agent and a second nuclease agent to produce a first digested nucleic acid, wherein the first nuclease agent generates a nick on a first strand of the first nucleic acid at a first target site, and the second nuclease agent generates a nick on a second strand of the first nucleic acid at a second target site, to produce the first digested nucleic acid comprising 5 Or 3' overhanging sequence at one of its ends;

(b) annealing the first digested nucleic acid and a second nucleic acid comprising a complementary sequence to the 5 Or 3' overhanging sequence; and

(c) ligating the first digested nucleic acid and the second nucleic acid.

11. The method of claim 10, wherein step (b) further comprises extending the 3' end of the first strand using the second strand as a template and extending the 3' end of the second strand using the first strand as a template.

12. The method of claim 10 or 11, wherein at least one of the first nuclease agent and the second nuclease agent comprises a Cas9 protein and a guide RNA(gRNA) (gRNA- Cas complex) that targets the first target site or the second target site.

13. The method of claim 12, wherein the Cas9 protein comprises a RuvC domain and a HNH domain, one of which lacks endonuclease activity.

14. The method of any one of claims 10-13, wherein the first target site is separated by at least 4 bp from the second target site.

15. The method of any one of claims 10-14, wherein the gRNA comprises a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), and wherein at least one of the first target site and the second target site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

16. A method for assembling two or more nucleic acids, comprising:

(a) contacting a first nucleic acid with at least one nuclease agent to g a first digested nucleic acid;

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

wherein the joiner oligo 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 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.

17. The method of claim 16, wherein assembling in step (c) comprises: (i) annealing the first complementary sequence of the joiner oligo to the first digested nucleic acid and the second complementary sequence of the joiner oligo to the second nucleic acid; and

(ii) ligating the joiner oligo to the first digested nucleic acid and the second nucleic acid.

18. The method of claim 16 or 17, wherein the first complementary sequence and the second complementary sequence of the joiner oligo comprise between 15 and 120 complementary bases.

19. The method of any one of claims 16-18, wherein the spacer of the joiner oligo comprises non-complementary nucleic acids.

20. The method of any one of claims 16-18, wherein the first digested nucleic acid is seamlessly assembled to the second nucleic acid.

21. The method of claim 20, wherein the at least one nuclease agent is designed to cleave an at least 20 bp fragment from the end of the first nucleic acid at which the seamless assembly will occur,

wherein the spacer of the joiner oligo comprises a sequence identical to the at least 20 bp fragment, wherein no nucleic acid bases are present between the first complementary sequence and the at least 20 bp fragment, and no nucleic acid bases are present between the second complementary sequence and the at least 20 bp fragment,

such that assembly of the first nucleic acid with the joiner oligo and the second nucleic acid reconstitutes the at least 20 bp fragment and seamlessly assembles the first nucleic acid and the second nucleic acid.

22. The method of claim 20, wherein the at least one nuclease agent is designed to cleave an at least 20 bp fragment from the end of the second nucleic acid at which the seamless assembly will occur,

wherein the spacer of the joiner oligo comprises a sequence identical to the at least 20 bp fragment, wherein no nucleic acid bases are present between the first complementary sequence and the at least 20 bp fragment, and no nucleic acid bases are present between the second complementary sequence and the at least 20 bp fragment,

such that assembly of the first nucleic acid with the joiner oligo and the second nucleic acid reconstitutes the at least 20 bp fragment and seamlessly assembles the first nucleic acid and the second nucleic acid.

23. The method of claim 21 or 22, wherein the spacer comprises from about 20 bp to about 120 bp.

24. The method of any one of claims 16-23, wherein step (a) further comprises contacting the second nucleic acid with a second nuclease agent and an exonuclease, wherein the second nuclease agent cleaves the second nucleic acid to produce a second digested nucleic acid comprising a nucleotide sequence that is complementary to the second

complementary sequence of the joiner oligo, wherein the first digested nucleic acid is assembled to the second digested nucleic acid.

25. The method of any one of claims 16-23, wherein step (a) further comprises contacting the second nucleic acid with a restriction enzyme or meganuclease and an exonuclease, wherein the restriction enzyme or meganuclease cleaves the second nucleic acid to produce a second digested nucleic acid comprising a nucleotide sequence that is complementary to the second complementary sequence in the joiner oligo, wherein the first digested nucleic acid is assembled to the second digested nucleic acid.

26. The method of claim 24 or 25, wherein step (b) further comprises extending the 3' end of the first digested nucleic acid and/or the second digested nucleic acid.

27. The method of any one of claims 16-26, wherein the joiner oligo is assembled to the first nucleic acid and the second nucleic acid in the same reaction.

28. The method of any one of claims 16-26, wherein the joiner oligo is assembled to the first nucleic acid and the second nucleic acid sequentially.

29. The method of any one of claims 24-28, wherein the at least one nuclease agent and/or the second nuclease agent comprises a Cas protein and a guide RNA (gRNA) (gRNA-Cas complex), a zinc finger nuclease, or a Transcription Activator-Like Effector Nuclease (TALEN) that targets the first or the second target site.

30. The method of claim 29, wherein at least one of the first nuclease agent and the second nuclease agent comprises the Cas protein and the guide RNA (gRNA) (gRNA- Cas complex),

wherein the Cas protein is a Cas9 protein, the gRNA comprises a nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), and at least one of the first target site and the second target site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

31. The method of claim 30, wherein the Cas9 protein comprises a RuvC domain and a HNH domain, at least one of which lacks endonuclease activity.

32. The method of any one of claims 10-31, wherein the first nucleic acid, the second nucleic acid, or both nucleic acids are derived from a bacterial artificial chromosome.

33. The method of any one of claims 10-32, wherein the first nucleic acid, the second nucleic acid, or both nucleic acids comprise a human DNA, a rodent DNA, a synthetic DNA, or a combination thereof.

34. The method of any one of claims 10-33, wherein the first nucleic acid, the second nucleic acid, or both nucleic acids are at least 10 kb.

35. The method of any one of claims 16-34, wherein the joiner oligo comprises a linear double stranded DNA fragment.

36. The method of claim 35, wherein the linear double stranded DNA fragment does not comprise a selection cassette.

37. A method for assembling two or more nucleic acids, comprising:

(a) contacting a first nucleic acid with at least one nuclease agent to generate a first digested nucleic acid; (b) contacting a second nucleic acid with a second nuclease agent to generate a second digested nucleic acid;

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

wherein the joiner oligo 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 digested nucleic acid;

wherein the exonuclease exposes the first and second complementary sequences; and

(d) assembling the joiner oligo with the first digested nucleic acid and the second digested nucleic acid.