Modular universal plasmid design strategy for the assembly and editing of multiple DNA constructs for multiple hosts

Patent metadata

Field	Value
Publication number	US-11098305-B2
Application number	US-201916535245-A
Country	US
Kind code	B2
Filing date	Aug 8, 2019
Priority date	Feb 10, 2017
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

Methods, compositions, and kits for high throughput DNA assembly reactions in vitro. Modular CRISPR DNA constructs comprising modular insert DNA parts flanked by cloning tag segments comprising pre-validated CRISPR protospacer/protospacer adjacent motif sequence combinations.

First claim

What is claimed is:

1. A method for preparing a recombinant nucleic acid molecule, the method comprising:
A) incubating a mixture comprising:
i) a plurality of different DNA inserts, wherein each DNA insert is flanked by two cloning tags (cTAGs), each cTAG comprising:
a) one or more validated CRISPR landing site(s), each comprising a protospacer sequence operably linked to a protospacer adjacent motif (PAM); wherein each cTAG was artificially introduced into each of the DNA inserts; and

ii) one or more CRISPR complexes targeting at least one of the cTAGs present in at least two of the plurality of DNA inserts, each CRISPR complex comprising:
a) a CRISPR endonuclease, and
b) a guide RNA capable of recruiting said CRISPR endonuclease to one of said targeted cTAGs;

under conditions which allow for digestion of the targeted cTAG(s) in at least two of the plurality of DNA inserts to generate digested DNA ends, and

B) incubating the DNA inserts with digested DNA ends generated in (A) under conditions which allow for covalent joining of the digested DNA ends, thereby producing a recombinant nucleic acid molecule; wherein the recombinant nucleic acid molecule comprises the DNA inserts that are covalently joined in the method, wherein each covalently joined DNA insert is individually flanked by the cTAGs that were flanking the DNA insert before covalent joining.

2. The method of claim 1 , wherein the digested DNA ends are sticky ends with overhang sequences capable of hybridizing to each other prior to the covalent joining of the digested ends.

3. The method of claim 2 , wherein the CRISPR endonuclease is Cpf1.

4. The method of claim 1 , wherein the digested DNA ends are blunt ends.

5. The method of claim 4 , wherein the blunt ends are further digested with a ssDNA exonuclease to create sticky ends with overhang sequences, and wherein step B) further comprises adding a bridging DNA sequence capable of hybridizing to the overhang sequences prior to the covalent joining of the digested ends.

6. The method of claim 1 , wherein the CRISPR endonuclease is Cas9.

7. The method of claim 1 , wherein the method further comprises the step of:
i) separating the digested cTAG sequences from the CRISPR complexes prior to step (B), and/or
ii) inactivating the CRISPR complexes prior to step (B).

8. The method of claim 7 , wherein the separation step comprises a DNA purification step.

9. The method of claim 7 , wherein the inactivation step comprises heat or chemical inactivation of said CRISPR complexes.

10. The method of claim 1 , wherein the two cTAGs of each of the plurality of DNA inserts form a cTAG pair, and wherein said cTAG pair is unique from all other cTAG pairs of the DNA inserts that are covalently joined in the method.

11. The method of claim 10 , wherein at least one of the cTAGs in each cTAG pair is the same as at least one other cTAG in a different cTAG pair.

12. A method for DNA sequence editing, said method comprising:
A) providing:
i) a CRISPR DNA construct comprising a CRISPR multi-clonal site, said multi-clonal site comprising at least two distinct cloning tags (cTAGs), said at least two distinct cloning tags having been artificially introduced into the CRISPR DNA construct, wherein each cTAG comprises:
a) one or more validated CRISPR landing site(s), each comprising a protospacer sequence operably linked to a protospacer adjacent motif (PAM); wherein at least one of said CRISPR landing sites is unique within the CRISPR DNA construct;

ii) a replacement DNA insert, wherein said replacement DNA insert is flanked by a first and second insert cTAG;
a) wherein the first insert cTAG comprises the validated CRISPR landing site(s) of one of the distinct cTAGs of the CRISPR DNA construct, and the second insert cTAG comprises the validated CRISPR landing site(s) of another distinct cTAG of the CRISPR DNA construct; and

iii) a first and second CRISPR complex targeting the first and second insert cTAGs, respectively, each CRISPR complex comprising:
a) a CRISPR endonuclease, and
b) a guide RNA capable of recruiting said CRISPR endonuclease to one of said targeted insert cTAGs;

wherein the CRISPR DNA construct and replacement DNA insert defined in (i) and (ii) are each incubated with the first and second CRISPR complexes defined in (iii) in a single reaction or separate reactions; wherein the first and second CRISPR complexes cleave the first and second insert cTAGs and their corresponding distinct cTAGs to generate digested DNA ends, and

B) incubating the replacement DNA insert and CRISPR DNA construct with digested DNA ends generated in step (A) under conditions which allow for covalent joining of the digested DNA ends, thereby producing an edited CRISPR DNA construct; wherein the edited CRISPR DNA construct comprises the cTAG sequences of the replacement DNA insert that is covalently joined by the method, and wherein the cTAGS of the replacement DNA insert are flanking the replacement DNA insert after covalent joining.

13. The method for DNA sequence editing of claim 12 , wherein the incubation of step (B) comprises a ligase.

14. The method of claim 12 , wherein the method further comprises the step of:
i) separating the cleaved first and second insert cTAGs and their corresponding distinct cTAGs from the CRISPR complexes prior to step (B), and/or
ii) inactivating the CRISPR complexes prior to step (B).

15. The method of claim 14 , wherein the separation step comprises a DNA purification step.

16. The method of claim 14 , wherein the inactivation step comprises heat or chemical inactivation of said CRISPR complexes.

17. The method of claim 12 , wherein the digested DNA ends are sticky ends with overhang sequences capable of hybridizing to each other prior to the covalent joining of the digested ends.

18. The method of claim 12 , wherein the CRISPR endonuclease is Cpf1.

19. The method of claim 12 , wherein the digested DNA ends are blunt ends.

20. The method of claim 19 , wherein the blunt ends are further digested with a ssDNA exonuclease to create sticky ends with overhang sequences, and wherein step B) further comprises adding a bridging DNA sequence capable of hybridizing to the overhang sequences prior to the covalent joining of the digested ends.

21. The method of claim 12 , wherein the CRISPR endonuclease is Cas9.

Assignees

• Zymergen Inc

Inventors

• Chaikind Brian
• Van Rossum Hendrik Marinus

Classifications

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title