Increased nucleic-acid guided cell editing in yeast

Abstract

The present disclosure provides methods to increase the percentage of edited yeast cells in a cell population using nucleic-acid guided editing, and automated multi-module instruments for performing these methods.

We claim: 
     
         1 . A library of editing vectors for performing nucleic acid-guided nuclease editing in yeast, where at least one vector in the library of vectors comprises:
 a yeast backbone;   an origin of replication;   a first non-minimal or non-core constitutive or inducible promoter driving transcription of an editing cassette followed by a terminator element 3′ to the editing cassette, wherein each editing cassette comprises a gRNA sequence and donor DNA sequence;   a second non-minimal or non-core constitutive promoter driving transcription for a degron-survival marker fusion gene followed by a terminator element 3′ to the degron-survival marker fusion gene;   a third non-minimal constitutive or inducible promoter driving transcription of a nuclease coding sequence with a terminator element 5′ to the nuclease or nuclease fusion coding sequence.   
     
     
         2 . The library of editing vectors of  claim 1 , wherein the library of editing vectors comprises a collection of vectors where editing cassettes with different donor DNA sequences are associated with a different barcode. 
     
     
         3 . The library of editing vectors of  claim 1 , wherein the library is isothermally assembled. 
     
     
         4 . The library of editing vectors of  claim 1 , wherein the minimal promoter is located between −35 to +35 of the transcription start site. 
     
     
         5 . The library of editing vectors of  claim 1 , wherein the degron survival marker is an antibiotic. 
     
     
         6 . The library of editing vectors of  claim 5 , wherein the antibiotic is selected from the group consisting of hygromycin, blasticidin, kanamycin, and nourseothricin. 
     
     
         7 . The library of editing vectors of  claim 1 , wherein the degron-survival marker fusion gene is an ubiquitin-dependent degron. 
     
     
         8 . The library of editing vectors of  claim 7 , wherein the ubiquitin-dependent degron is selected from Ura3-d degron, Ubi-R degron, Ubi-M degron, Ubi-Q degron, Ubi-E degron, ZF1 degron, C-terminal phosphodegron; Ts-degron; lt-degron; auxin inducible degron; DD-degron, LID-degron; PSD degron, B-LID degron; and a TIPI degron. 
     
     
         9 . The library of editing vectors of  claim 7 , wherein the ubiquitin-dependent degron is a Ura3-d degron. 
     
     
         10 . The library of editing vectors of  claim 7 , wherein the first, second, and third non-minimal or non-core constitutive or inducible promoters are the same. 
     
     
         11 . The library of editing vectors of  claim 7 , wherein the first, second, and third non-minimal or non-core constitutive or inducible promoters are different. 
     
     
         12 . A library of editing vectors for performing nucleic acid-guided nuclease editing in yeast, where at least one vector in the library of vectors comprises:
 a yeast backbone;   an origin of replication;   a first non-minimal or non-core constitutive or inducible promoter driving transcription of an editing cassette followed by a terminator element 3′ to the editing cassette, wherein each editing cassette comprises a gRNA sequence and donor DNA sequence;   a minimal promoter or a weak constitutive promoter driving transcription of a coding sequence for a survival marker gene followed by a terminator element 3′ to the survival marker gene;   a second non-minimal or non-core constitutive or inducible promoter driving transcription of a nuclease coding sequence with a terminator element 5′ to the nuclease or nuclease fusion coding sequence.   
     
     
         13 . The library of editing vectors of  claim 12 , wherein the library of editing vectors comprises a collection of vectors where editing cassettes with different donor DNA sequences are associated with a different barcode. 
     
     
         14 . The library of editing vectors of claim  122 , wherein the library is isothermally assembled. 
     
     
         15 . The library of editing vectors of claim  122 , wherein the minimal promoter is located between −35 to +35 of the transcription start site. 
     
     
         16 . The library of editing vectors of claim  122 , wherein the minimal promoter is selected from the group consisting of any one of SEQ ID Nos: 1-25. 
     
     
         17 . The library of editing vectors of claim  122 , wherein the weak constitutive promoter is selected from the group consisting of any one of SEQ ID Nos: 26-30. 
     
     
         18 . The library of editing vectors of  claim 12 , wherein the survival marker gene is an antibiotic. 
     
     
         19 . The library of editing vectors of  claim 18 , wherein the antibiotic is selected from the group consisting of hygromycin, blasticidin, kanamycin, and nourseothricin. 
     
     
         20 . The library of editing vectors of  claim 12 , wherein the first and second non-minimal or non-core constitutive or inducible promoters are the same. 
     
     
         21 . The library of editing vectors of  claim 12 , wherein the first and second non-minimal or non-core constitutive or inducible promoters are different.