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US 2021/0207149 A1Published application
Increased nucleic acid-guided cell editing via a lexa-rad51 fusion protein
AssigneeInscripta, Inc.
InventorsMiles Gander, Tian Tian, Eileen Spindler
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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.
Claims23
A method for increasing total edit fraction by CRISPR editing in yeast cells to greater than 80% comprising the steps of:
- providing a first set of editing vectors for nucleic acid-guided nuclease editing in yeast, wherein the first set of editing vectors comprises:
- a promoter driving transcription of a first set of editing cassettes, wherein each editing cassette in the first set of editing cassettes comprises 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 MAD7;
- 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;
- providing a population of yeast cells;
- making the population of yeast cells electrocompetent;
- transforming the first set of editing vectors into the yeast cells;
- allowing the transformed yeast cells to recover;
- providing conditions to allow for nucleic acid-guided editing in the selected yeast cells to produce edited yeast cells;
- and
- growing the edited yeast cells to a stationary phase of growth.
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Dependencies are inferred from the claim text.