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US 11,306,299 B2Granted
Simultaneous multiplex genome editing in yeast
AssigneeInscripta, Inc.
InventorsSkylar Stefani, Tian Tian, Miles Gander
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Abstract
The present disclosure provides compositions of matter, methods and instruments for editing nucleic acids in live yeast cells.
Claims30
A library of linear vector backbones and a library of editing cassettes to be transformed into yeast cells comprising:
- a first linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a first antibiotic resistance gene, and a yeast origin of replication;
- a second linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a second antibiotic resistance gene, and a yeast origin of replication;
- and
- a library of editing cassette constructs, wherein each editing cassette construct comprises from 5′ to 3′:
- a pol II promoter;
- a transcription start site;
- a first editing cassette wherein the first editing cassette comprises a coding sequence for a first gRNA and a coding sequence for a first donor DNA, wherein the first donor DNA comprises a rational, desired edit to a first target sequence and an edit configured to render inactive a first proto-spacer motif (PAM) in the first target sequence;
- a tRNA linker;
- a second editing cassette wherein the second editing cassette comprises a coding sequence for a second gRNA and a coding sequence for a second donor DNA, wherein the second donor DNA comprises a rational, desired edit to a second target sequence and an edit configured to render inactive a second proto-spacer motif (PAM) in the second target sequence;
- a coding sequence for a self-cleaving ribozyme;
- and a pol II terminator;
- wherein the first and second editing cassettes in the library of editing cassette constructs are different among editing cassettes constructs, and wherein homology exists between the library of editing cassette constructs and the first and second linear vector backbones.
9 more elements
A library of linear vector backbones and a library of editing cassettes to be transformed into yeast cells comprising:
- a first linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a first antibiotic resistance gene, and a yeast origin of replication;
- a second linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a second antibiotic resistance gene, and a yeast origin of replication;
- a third linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a third antibiotic resistance gene, and a yeast origin of replication;
- a library of editing cassette constructs, wherein each editing cassette construct comprises from 5′ to 3′:
- a pol II promoter;
- a transcription start site;
- a first editing cassette wherein the first editing cassette comprises a coding sequence for a first gRNA and a coding sequence for a first donor DNA, wherein the first donor DNA comprises a rational, desired edit to a first target sequence and an edit configured to render inactive a first proto-spacer motif (PAM) in the first target sequence;
- a tRNA linker;
- a second editing cassette wherein the second editing cassette comprises a coding sequence for a second gRNA and a coding sequence for a second donor DNA, wherein the second donor DNA comprises a rational, desired edit to a second target sequence and an edit configured to render inactive a second proto-spacer motif (PAM) in the second target sequence;
- a coding sequence for a self-cleaving ribozyme;
- and a pol II terminator;
- wherein the first and second editing cassettes in the library of editing cassette constructs are different among editing cassettes constructs, and wherein homology exists between the library of editing cassette constructs and the first, second and third linear vector backbones.
9 more elements
Dependencies are inferred from the claim text.