DNA ASSEMBLY USING AN RNA-PROGRAMMABLE NICKASE

• US 20150031089A1
• Filed: 06/30/2014
• Published: 01/29/2015
• Est. Priority Date: 07/29/2013
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Lindstrom, Derek Lee
• Granted Patent: US 10,421,957 B2
• Time in Patent Office: Days
• US Class Current: 435/91.52
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

This disclosure provides, among other things, a method of combining nucleic acid fragments, comprising: (a) providing two double-stranded DNA molecules with a common sequence, wherein the common sequence is at the end of each molecule; (b) nicking one strand in the common sequence of both molecules at a respective nicked site; (c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to one end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; (d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules. Alternative ways for performing the method are also provided.

Claims

1. A method of combining nucleic acid fragments, comprising:

(a) providing two double-stranded DNA molecules having a common sequence, wherein the common sequence is at the end of each molecule;

(b) nicking one strand in the common sequence of both molecules at a respective nicked site;

(c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to said end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; and

(d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules.

2. The method of claim 1, wherein the overhanging sequences of (c) are in the range of 4 to 30 bases in length.

3. The method of claim 1, wherein the overhanging sequences of (c) are in the range of 18-23 bases in length.

4. The method of claim 1, wherein the denaturing is by raising the temperature to a temperature in the range of 60°

C. to 90°

C.

5. The method of claim 1, wherein the molecules are ligated in step (d) using a thermostable ligase.

6. The method of claim 5, wherein the method comprises repeating steps (c) and (d) multiple times.

7. The method of claim 1, wherein the first and second molecules are each of at least 100 bp in length.

8. The method of claim 1, wherein the Cas9 nicking enzyme has amino acid substitutions at D10 or H840, or a site corresponding thereto.

9. The method of claim 1, wherein the nicking step (b) is done using a Cas9 nickase and guide RNAs that are complementary to opposite strands of the common sequence.

10. The method of claim 1, wherein the product of step (d) comprises a coding sequence.

11. The method of claim 1, wherein steps (b) to (d) are done in a single vessel and no additional reagents are added between steps (b) to (d).

12. A method for recombining DNA molecules, comprising:

(a) obtaining a first double-stranded DNA molecule of formula A and a second double-stranded DNA molecule of formula B;

(b) nicking;

(i) the first molecule of double-stranded DNA at a site that is 4-30 bases from the 3′

end of one of the strands of the first molecule, using Cas9 nickase and a first guide RNA;

(ii) the second molecule of double-stranded DNA at a site that is 4-30 bases from the 5′

end of one of the strands of the second molecule, using Cas9 nickase and a second guide RNA;

(c) denaturing the nicked products of (b) in moderately denaturing conditions, thereby producing double-stranded products that comprise overhangs in range of 10-30 bases in length; and

(d) ligating the double-stranded products of (c) by annealing the overhangs to a splint oligonucleotide and ligating the ends of the products using a ligase, thereby joining the double-stranded products to the splint oligonucleotide and producing a product molecule of the formula A-B.

13. The method of claim 12, wherein the overhanging sequences of (c) are in the range of 18-23 bases in length.

14. The method of claim 12, wherein the denaturing is by raising the temperature to a temperature in the range of 60°

C. to 90°

C.

15. The method of claim 12, wherein the ligase used in step (d) is a thermostable ligase.

16. The method of claim 15, wherein the method comprises repeating steps (c) and (d) multiple times.

17. The method of claim 12, wherein the first and second molecules are each of at least 100 bp in length.

18. The method of claim 12, wherein the Cas9 nicking enzyme has amino acid substitutions at D10 or H840, or a site corresponding thereto.

19. The method of claim 12, wherein the nicking step (b) is done using a Cas9 nickase and guide RNAs that are complementary to opposite strands of the common sequence.

20. The method of claim 12, wherein steps (b) to (d) are done in a single vessel and no additional reagents are added between steps (b) to (d).