Increased nucleic acid-guided cell editing via a lexa-rad51 fusion protein

Patent metadata

Field	Value
Publication number	US-2021010006-A1
Application number	US-202016917905-A
Country	US
Kind code	A1
Filing date	Jul 1, 2020
Priority date	Jul 8, 2019
Publication date	Jan 14, 2021
Grant date	—

Abstract

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

First claim

1 . An editing vector for nucleic acid-guided nuclease editing in yeast comprising:
a promoter driving transcription of an editing cassette comprising a guide nucleic acid and a donor DNA sequence; a yeast origin of replication; a bacterial origin of replication; a promoter driving transcription of a coding sequence for a nuclease; a promoter driving transcription of a selection marker; one or more LexA DNA binding sites; and a promoter driving transcription of a LexA-linker-Rad51 fusion protein.

2 . The editing vector of claim 1 , wherein the LexA-linker-Rad51 fusion protein comprises a portion of a LexA protein and a portion of a Rad51 protein.

3 . The editing vector of claim 2 , wherein the portion of a LexA protein comprises SEQ ID No. 1.

4 . The editing vector of claim 2 , wherein the portion of a Rad51 protein comprises SEQ ID No. 2.

5 . The editing vector of claim 1 , wherein the linker of the LexA-linker-Rad51 fusion protein comprises a polyglycine linker or a glycine-serine linker.

6 . The editing vector of claim 1 , wherein the one or more LexA DNA binding sites comprise SEQ ID No. 3.

7 . The editing vector of claim 1 , wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is an yeast alcohol dehydrogenase 1 promoter, a pGPD promoter, a pTEF1 promoter, a pACT1 promoter, a pRNR2 promoter, a pCYC1 promoter, a pTEF2 promoter, a pHXT7 promoter, a pYEF3 promoter, a pRPL3 promoter, a pRPL4 promoter or a pGAL1 promoter.

8 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the yeast alcohol dehydrogenase 1 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein an ADH1 terminator element.

9 . wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pGDP promoter; and
the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein GDP terminator element.

10 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pGDP promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein GDP terminator element.

11 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pTEF1 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein TEF1 terminator element.

12 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pTEF2 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein TEF2 terminator element.

13 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pACT1 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein ACT1 terminator element.

14 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pRNR2 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein RNR2 terminator element.

15 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pCYC1 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein CYC1 terminator element.

16 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pHXT7 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein HXT7 terminator element.

17 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pYEF3 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein YEF3 terminator element.

18 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pRPL3 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein RPL3 terminator element.

19 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pRPL4 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein RPL4 terminator element.

20 . The editing vector of claim 7 ,
wherein the promoter driving transcription of the LexA-linker-Rad51 fusion protein is the pGAL1 promoter; and the editing vector further comprises 3′ to the LexA-linker-Rad51 fusion protein GAL1 terminator element.

Assignees

• Inscripta Inc

Inventors

• Gander Miles
• Tian Tian
• Spindler Eileen

Classifications

• C12N15/81 Primary

  for yeasts · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/62 Primary

  DNA sequences coding for fusion proteins · CPC title

• C12N2310/20

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

• C07K14/47

  from mammals · CPC title