# US 11,306,299 B2 · Simultaneous multiplex genome editing in yeast > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,299 B2 - Document ID: US11306299B2 - Status: Granted - Kind: B2 - Application number: 17/518,556 - Filed: 2021-11-03 - Published: 2022-02-24 - Granted: 2022-04-19 - Earliest claimed: 2019-03-25 - Assignees: Inscripta, Inc. - Applicants: Inscripta, Inc. - Inventors: Skylar Stefani; Tian Tian; Miles Gander - Web page: https://patentgrep.com/patent/US11306299B2 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes} - C12N 15/102: {Mutagenizing nucleic acids} - 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)} - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12N 15/81: for yeasts - C12N 15/85: for animal cells - C12Q 1/44: involving esterase - C12N 15/90: Stable introduction of foreign DNA into chromosome - C12N 2310/128: processing or releasing ribozyme - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present disclosure provides compositions of matter, methods and instruments for editing nucleic acids in live yeast cells. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1 further comprising 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, and wherein homology exists between the library of editing cassettes and the third linear vector backbones. ### Claim 3 (depends on 2) The libraries of linear vector backbones and editing cassette constructs of claim 2 further comprising a fourth linear vector backbone comprising a coding sequence for a nuclease, a coding sequence for a fourth antibiotic resistance gene, and a yeast origin of replication, and wherein homology exists between the library of editing cassettes and the fourth linear vector backbones. ### Claim 4 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1 wherein the coding sequence for the nuclease in the first and second linear vector backbones is the coding sequence for the same nuclease. ### Claim 5 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the first antibiotic resistance gene confers resistance to hygromycin and the second antibiotic resistance gene confers resistance to G418. ### Claim 6 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the first and second linear vector backbones comprise the pol II promoter driving expression of the editing cassette construct. ### Claim 7 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein each linear vector backbone further comprises an origin of replication functional in bacteria. ### Claim 8 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in a hepatitis delta virus (HDV)-like ribozyme family, a self-cleaving ribozyme in a glucosamine-6-phosphate synthase ribozyme family, a self-cleaving ribozyme in a hammerhead ribozyme family, a self-cleaving ribozyme in a hairpin ribozyme family, a self-cleaving ribozyme in a Neurospora Varkud satellite ribozyme family, a self-cleaving ribozyme in a twister ribozyme family, a self-cleaving ribozyme in a twister sister ribozyme family, a self-cleaving ribozyme in a hatchet ribozyme family, or a self-cleaving ribozyme in a pistol ribozyme family. ### Claim 9 (depends on 8) The libraries of linear vector backbones and editing cassette constructs of claim 8, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in the hepatitis delta virus (HDV)-like ribozyme family. ### Claim 10 (depends on 8) The libraries of linear vector backbones and editing cassette constructs of claim 8, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in the glucosamine-6-phosphate synthase ribozyme family. ### Claim 11 (depends on 8) The libraries of linear vector backbones and editing cassette constructs of claim 8, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in the Neurospora Varkud satellite ribozyme family. ### Claim 12 (depends on 8) The libraries of linear vector backbones and editing cassette constructs of claim 8, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in the twister ribozyme family. ### Claim 13 (depends on 8) The libraries of linear vector backbones and editing cassette constructs of claim 8, wherein the self-cleaving ribozyme is a self-cleaving ribozyme in the twister sister ribozyme family. ### Claim 14 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, comprising a second self-cleaving ribozyme 3′ of the transcription start site. ### Claim 15 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the pol II promoter is a cell-type specific promoter, a tissue-specific promoter, or a synthetic promoter. ### Claim 16 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the pol II promoter is a constitutive fungal promoter. ### Claim 17 (depends on 16) The libraries of linear vector backbones and editing cassette constructs of claim 16, wherein the constitutive fungal pol II promoter is a pPGK1, pTDH3, pENO2, pADH1, pTPI1, pTEF1, pTEF2, pYEF3, pRPL3, pRPL15A, pRPL4, pRPL8B, pSSA1, pSSB1, pCYC1, or pPDA1 promoter. ### Claim 18 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the tRNA linker is an alanine tRNA. ### Claim 19 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the tRNA linker is a glycine tRNA. ### Claim 20 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the tRNA linker is a threonine tRNA. ### Claim 21 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the pol II promoter is a constitutive mammalian promoter. ### Claim 22 (depends on 21) The libraries of linear vector backbones and editing cassette constructs of claim 21, wherein the pol II promoter is a pCMV, pEF1a, pSV40, pPGK1, pUbc, human beta actin promoter, or pCAG promoter. ### Claim 23 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the pol II promoter is an inducible promoter. ### Claim 24 (depends on 23) The libraries of linear vector backbones and editing cassette constructs of claim 23, wherein an inducible promoter is a PHO5 promoter, a MET3 promoter, a CUP1 promoter, GAL1 promoter, or a GEV or LEV promoter system. ### Claim 25 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the first gRNA in the first editing cassette is 5′ of the first donor DNA and wherein the second gRNA in the second editing cassette is 5′ of the second donor DNA. ### Claim 26 (depends on 1) The libraries of linear vector backbones and editing cassette constructs of claim 1, wherein the first gRNA in the first editing cassette is 3′ of the first donor DNA and wherein the second gRNA in the second editing cassette is 3′ of the second donor DNA. ### Claim 27 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. ### Claim 28 (depends on 27) The libraries of linear vector backbones and editing cassette constructs of claim 27, wherein the tRNA linker is an alanine tRNA. ### Claim 29 (depends on 27) The libraries of linear vector backbones and editing cassette constructs of claim 27, wherein the tRNA linker is a glycine tRNA. ### Claim 30 (depends on 27) The libraries of linear vector backbones and editing cassette constructs of claim 27, wherein the tRNA linker is a threonine tRNA.