Simultaneous multiplex genome editing in yeast

Patent metadata

Field	Value
Publication number	US-11274296-B2
Application number	US-202117463956-A
Country	US
Kind code	B2
Filing date	Sep 1, 2021
Priority date	Mar 25, 2019
Publication date	Mar 15, 2022
Grant date	Mar 15, 2022

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 composition of matter comprising libraries of linear vector backbones and libraries of editing cassettes to be transformed into yeast cells comprising:
a first linear vector backbone library, wherein each first linear vector backbone in the first linear vector backbone library comprises a coding sequence for a nuclease, a coding sequence for a first antibiotic resistance gene, first homology regions for inserting a first library of editing cassettes, and a 2μ origin of replication;
a second linear vector backbone library, wherein each second linear vector backbone in the second linear vector backbone library comprises a coding sequence for a nuclease, a coding sequence for a second antibiotic resistance gene, second homology regions for inserting a second library of editing cassettes, and a 2μ origin of replication;
the first library of editing cassettes, wherein each editing cassette in the first library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the first library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between the editing cassettes in the first library of editing cassettes and the first linear vector backbone library; and
the second library of editing cassettes, wherein each editing cassette in the second library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the second library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between the editing cassettes in the second library of editing cassettes and the second linear vector backbone library.

2. The composition of matter of claim 1 further comprising a third linear vector backbone library wherein each third linear vector backbone in the third linear vector backbone library comprises a coding sequence for a nuclease, third homology regions for inserting a third library of editing cassettes, a coding sequence for a third antibiotic resistance gene, and a 2μ origin of replication; and the third library of editing cassettes, wherein each editing cassette in the third library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the third library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between editing cassettes in the third library of editing cassettes and the third linear vector backbone library.

3. The composition of matter of claim 2 further comprising a fourth linear vector backbone library wherein each fourth linear vector backbone in the fourth linear vector backbone library comprises a coding sequence for a nuclease, fourth homology regions for inserting a fourth library of editing cassettes, a coding sequence for a fourth antibiotic resistance gene, and a 2μ origin of replication; and the fourth library of editing cassettes, wherein each editing cassette in the fourth library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the fourth library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between the editing cassettes in the fourth library of editing cassettes and the fourth linear vector backbone library.

4. The composition of matter of claim 1 , wherein the first and second antibiotic resistance genes confer resistance to hygromycin, G418, blasticidin, puromycin or nourseothricin and the first and second antibiotic resistance genes are different.

5. The composition of matter of claim 1 , wherein at least one editing cassette of each library of editing cassettes comprises two gRNAs and two donor DNAs.

6. The composition of matter of claim 1 , wherein each editing cassette of each library of editing cassettes comprises three gRNAs and three donor DNAs.

7. The composition of matter of claim 1 , where at least one linear vector backbone in each linear backbone library further comprises a promoter for driving expression of the editing cassette.

8. The composition of matter of claim 7 , wherein the promoter is a pol II promoter.

9. The composition of matter of claim 8 , wherein the pol II promoter is a pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2, pYEF3, pRPL3, pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, or pPDA1 promoter.

10. The composition of matter of claim 1 , wherein each linear vector backbone in each linear backbone library further comprises an origin of replication functional in bacteria.

11. A composition of matter comprising libraries of linear vector backbones and libraries of editing cassettes to be transformed into yeast cells comprising:
a first linear vector backbone library wherein each linear vector backbone in the first linear vector backbone library comprises 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, first homology regions for inserting a first library of editing cassettes, and a 2μ origin of replication;
a second linear vector backbone library wherein each linear vector backbone in the second linear vector backbone library comprises 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, second homology regions for inserting a second library of editing cassettes, and a 2μ origin of replication;
the first library of editing cassettes, wherein each editing cassette in the first library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the first library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between the first library of editing cassettes and the first linear vector backbone library; and
the second library of editing cassettes, wherein each editing cassette in the second library of editing cassettes comprises a gRNA and a donor DNA, wherein the gRNAs and donor DNAs of different editing cassettes in the second library of editing cassettes target different target regions in a yeast genome, wherein each donor DNA comprises an edit configured to render inactive a protospacer motif (PAM) in the target sequence, and wherein homology exists between the second library of editing cassettes and the second linear vector backbone library.

12. The composition of matter of claim 11 further comprising a third linear vector backbone library wherein each third linear vector backbone in the third linear vector backbone library comprises a coding sequence for a nuclease, third homology regions for inserting a third library of editing cassettes, a coding sequence for a first portion of a second antibiotic resistance gene fused to the N-terminus of a second intein, and a 2μ origin of replication; a fourth linear vector backbone library wherein each fourth linear vector backbone in the fourth

Assignees

• Inscripta Inc

Inventors

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

Classifications

• C12N2310/20

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

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12N15/1093

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

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/1037 Primary

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