Simultaneous multiplex genome editing in yeast

Patent metadata

Field	Value
Publication number	US-11746347-B2
Application number	US-202217679203-A
Country	US
Kind code	B2
Filing date	Feb 24, 2022
Priority date	Mar 25, 2019
Publication date	Sep 5, 2023
Grant date	Sep 5, 2023

Abstract

The present disclosure provides compositions of matter, methods and instruments for editing nucleic acids in live yeast cells.

First claim

We claim:

1. A method for RNA-directed nuclease editing comprising the steps of:
designing and synthesizing a first linear vector backbone library comprising a coding sequence for a nuclease, a coding sequence for a first portion of a first antibiotic resistance gene fused to an N-terminus of a first intein, and first homology regions for inserting a first library of editing cassettes;
designing and synthesizing a second linear vector backbone library comprising a coding sequence for a nuclease, a coding sequence for a second portion of a first antibiotic resistance gene fused to an C-terminus of the first intein, and second homology regions for inserting a second library of editing cassettes;
designing and synthesizing the first library of editing cassettes, wherein each editing cassette comprises (i) a sequencing encoding a gRNA (ii) and a donor DNA, and wherein homology exists between the first library of editing cassettes and the first linear vector library;
designing and synthesizing the second library of editing cassettes, wherein each editing cassette comprises (i) a sequence encoding a gRNA and (ii) a donor DNA, and wherein homology exists between the second library of editing cassettes and the second linear vector library;
transforming cells with the first and second linear vector backbone libraries and first and second libraries of editing cassettes;
selecting for transformed cells by selecting for antibiotic resistance to the first antibiotic; and
providing conditions for RNA-directed nuclease editing in the cells to produce first edited cells.

2. The method of claim 1 , wherein the first intein is a Nostoc punctiforme PCC73102 split alpha subunit of the DNA polymerase III intein (NpuDnaE), a Synechocystis sp. PCC6803 DnaB helicase SspDnaB, or a CfaDnaE.

3. The method of claim 2 , wherein the first intein is the Nostoc punctiforme PCC73102 split alpha subunit of the DNA polymerase III intein (NpuDnaE).

4. The method of claim 2 , wherein the first intein is the Synechocystis sp. PCC6803 DnaB helicase SspDnaB.

5. The method of claim 2 , wherein the second first is the CfaDnaE.

6. The method of claim 1 , wherein the first antibiotic resistance gene confers resistance to hygromycin, G418, puromycin, blasticidin or nourseothricin.

7. The method of claim 1 , wherein some of the editing cassettes of the first and second libraries of editing cassettes comprise (i) two sequences encoding gRNAs and (ii) two donor DNAs.

8. The method of claim 7 , wherein some of the editing cassettes of the first and second libraries of editing cassettes comprise (iii) three sequences encoding gRNAs and (iii) three donor DNAs.

9. The method claim 1 , where each linear vector backbone in each linear backbone library further comprises a promoter driving expression of the editing cassette.

10. The method of claim 9 , wherein the promoter is a polymerase II (pol II) promoter.

11. The method of claim 10 , wherein the pol II promoter is a pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2, pYEF3, pRPL3, pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, or pPDA1 promoter.

12. The method of claim 1 , wherein each linear vector backbone in each first and second linear backbone library comprises an origin of replication functional in bacteria.

13. The method of claim 1 , wherein the cells are yeast cells.

14. The method of claim 13 , wherein the yeast cells are Saccharomyces cerevisiae cells.

15. The method of claim 1 , wherein at least one donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in a target sequence.

16. The method of claim 1 , wherein each linear vector backbone in each first and second linear backbone library comprises an origin of replication functional in yeast.

17. The method of claim 1 , further comprising the steps of:
designing and synthesizing a third linear vector backbone library comprising a coding sequence for a nuclease, third homology regions for inserting a third library of editing cassettes, and a coding sequence for a first portion of a second antibiotic resistance gene fused to the N-terminus of a second intein;
designing and synthesizing a fourth linear vector backbone library comprising a coding sequence for a nuclease, fourth homology regions for inserting a fourth library of editing cassettes, and a coding sequence for a second portion of a second antibiotic resistance gene fused to a C-terminus of the second intein;
designing and synthesizing the third library of editing cassettes, wherein each editing cassette comprises (i) sequence encoding a gRNA and (ii) a donor DNA, and wherein homology exists between the third library of editing cassettes and the third linear vector library;
designing and synthesizing the fourth library of editing cassettes, wherein each editing cassette comprises (i) sequence encoding a gRNA and (ii) a donor DNA, and wherein homology exists between the fourth library of editing cassettes and the fourth linear vector library;
transforming the first edited yeast cells with the third and fourth linear vector backbone libraries and third and fourth libraries of editing cassettes;
selecting for transformed first edited cells by selecting for antibiotic resistance to the second antibiotic; and
providing conditions for RNA-directed nuclease editing in the cells to produce second edited cells.

18. The method of claim 17 , wherein the second intein is a Nostoc punctiforme PCC73102 split alpha subunit of the DNA polymerase III intein (NpuDnaE), a Synechocystis sp. PCC6803 DnaB helicase SspDnaB, or a CfaDnaE.

19. The method of claim 18 , wherein the second intein is the Nostoc punctiforme PCC73102 split alpha subunit of the DNA polymerase III intein (NpuDnaE).

20. The method of claim 18 , wherein the second intein is the Synechocystis sp. PCC6803 DnaB helicase SspDnaB.

21. The method of claim 18 , wherein the second intein is the CfaDnaE.

22. The method of claim 17 , wherein the first and second antibiotic resistance genes confer resistance to hygromycin, G418, puromycin, blasticidin or nourseothricin and the first and second antibiotic resistance genes are different.

23. The method of claim 17 , wherein each linear vector backbone in each third and fourth linear backbone library comprises an origin of replication functional in bacteria.

24. The method of claim 17 , wherein each linear vector backbone in each third and fourth linear backbone library comprises a an origin of replication functional in yeast.

Assignees

• Inscripta Inc

Inventors

• Gander Miles
• Tian Tian
• Stefani Skylar
• Westfall Patrick

Classifications

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N2310/20

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

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title