SCARLESS DNA ASSEMBLY AND GENOME EDITING USING CRISPR/CPF1 AND DNA LIGASE

• US 20190330659A1
• Filed: 07/14/2017
• Published: 10/31/2019
• Est. Priority Date: 07/15/2016
• Status: Abandoned Application

• Current Assignee: Zymergen, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Zymergen, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: DeLoache, William C., Marinus van Rossum, Hendrik, Patel, Kedar Gautam
• Application Number:
  US16/310,895
• Publication Number:
  US 20190330659A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• 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 15/902 using homologous recombination
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The disclosure describes a scarless DNA assembly and genome editing methodology termed “CLIC” (CRISPR and Ligase Cloning), which utilizes a CRISPR/Cpf1 complex and DNA ligase to perform programmable gene editing and nucleotide assembly. The CLIC process is highly amenable to applications in vitro for the scarless assembly of a plurality of DNA parts simultaneously or in vivo for the site-specific insertion of one or more DNA molecules into the host genome.

Claims

1. A method for assembling gene constructs in vitro from a plurality of DNA fragments, said method comprising the steps of:

(a) providing a plurality of DNA fragments comprising a first and second DNA fragment, wherein said first DNA fragment comprises a sequence overlap of at least three nucleic acids anywhere within the second DNA fragment;

(b) digesting the first DNA fragment with a Cpf1 CRISPR system, thereby creating a sticky DNA end at the 5′ and

/or 3′

of said first DNA fragment, wherein said digested first DNA fragment ceases to be a target for said Cpf1 CRISPR system;

(c) annealing the sticky end of the digested first DNA fragment from step (b) to a second compatible sticky end on the second DNA fragment; and

(d) ligating the annealed DNA fragments from step (c) together, resulting in a ligated product;

wherein the resulting ligated product is an assembled construct;

wherein steps (b) and (d) are conducted in the same reaction without needing to inactivate the Cpf1 CRISPR system.

2. The method of claim 1, wherein no genetic scars are introduced into the assembled construct from practicing the method.

3. The method of claim 1, wherein the Cpf1 CRISPR system comprises i) a Cpf1 endonuclease, and ii) a crRNA capable of directing sequence-specific binding of the Cpf1 endonuclease to the first DNA fragment.

4. The method of claim 3, wherein the Cpf1 endonuclease is non-naturally occurring.

5. The method of claim 3, wherein the crRNA is non-naturally occurring.

6. The method of claim 1, wherein the Cpf1 CRISPR system of step (b) is targeted to a portion of the first DNA fragment that will be cleaved away from the first DNA fragment, such that the Cpf1 CRISPR system no longer targets the digested first DNA fragment.

9. The method of claim 1, wherein the provided second DNA fragment comprises a preexisting sticky end compatible with the sticky end of the digested first DNA fragment.

10. The method of claim 1, wherein step (b) further comprises digesting the second DNA fragment with a second Cpf1 CRISPR system, thereby creating the second compatible sticky end on the second DNA fragment, wherein said digested second DNA fragment ceases to be a target for said second Cpf1 CRISPR endonuclease system.

11. The method of claim 10, wherein the first Cpf1 CRISPR system and the second Cpf1 CRISPR system are identical.

7-8. -8. (canceled)

12. A method for editing the genome of a cell in vivo, said method comprising the steps of:

a) introducing into the cell a Cpf1 CRISPR system comprising one or more vectors comprising;

i) a first polynucleotide encoding a first crRNA that hybridizes to a first selected target sequence within the genome of the cell;

ii) a second polynucleotide encoding a second crRNA that hybridizes to a second selected target sequence within the genome of the cell; and

iii) a third polynucleotide encoding a Cpf1 endonuclease;

wherein components (i), (ii), and (iii) are expressed in the cell, and the Cpf1 endonuclease cleaves the cell'"'"'s genome at the first and second selected target sequences, thereby producing sticky ends on the cleaved ends of the cell'"'"'s genome;

wherein the first and second target sequences are positioned in an outwardly facing inverse orientation of a portion of the cell'"'"'s genome slated for removal, such that removal of said portion of the cell'"'"'s genome will also remove the first and second target sites from the genome;

(b) annealing the resulting genome sticky ends to each other; and

(c) ligating the annealed genome sticky ends from step (b).

13. The method of claim 12, wherein the one or more vectors of step (a) further comprise a fourth, insert polynucleotide, wherein said insert polynucleotide is also cleaved by the Cpf1 endonuclease, thereby creating sticky ends on the insert polynucleotide that are compatible with the sticky ends of the cell'"'"'s genome;

wherein the annealing step (b) is modified to anneal the sticky ends of the genome to the sticky ends of the insert polynucleotide; and

wherein the ligating step (c) is modified to ligate the annealed genome and insert sticky ends.

14. The method of claim 12, wherein no genetic scars are introduced into the genome from practicing the method.

15. The method of claim 13, wherein the fourth, insert polynucleotide, also comprises two copies of the first target sequence positioned in an inwardly facing inverse orientation, such that cleavage of said insert polynucleotide by the Cpf1 endonuclease removes the first and second copies of the first target site from the insert polynucleotide.

16. The method of claim 12, wherein the one or more vectors comprise a polynucleotide encoding a DNA ligase.

17. The method of claim 16, wherein the DNA ligase is selected from the group consisting of T4 ligase, and a T7 ligase.

18. The method of claim 12, wherein the Cpf1 endonuclease is non-naturally occurring.

19. The method of claim 12, wherein the first or second crRNA is non-naturally occurring.

20. The method of claim 16, wherein the DNA ligase is non-naturally occurring.

21. The method of claim 12, wherein the combination of (i), (ii), and (iii) is non-naturally occurring.

31. The method of claim 13, wherein no genetic scars are introduced into the genome from practicing the method.

22-30. -30. (canceled)