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US 10,837,021 B1Granted
Curing for recursive nucleic acid-guided cell editing
AssigneeInscripta, Inc.
InventorsTian Tian, Charles Johnson, Eileen Spindler
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Abstract
The present disclosure provides automated multi-module instrumentation and automated methods for performing recursive editing of live cells with curing of editing vectors from prior rounds of editing.
Claims20
A method for curing cells of choice during recursive nucleic acid-directed nuclease editing comprising:
- designing and synthesizing a first set of editing cassettes, wherein the first set of editing cassettes comprises one or more editing gRNA and donor DNA pairs wherein each editing gRNA and donor DNA pair is operably linked and under the control of a first inducible promoter;
- assembling the first set of editing cassettes into a vector backbone thereby forming a first set of editing vectors, wherein the vector backbone comprises a first selectable marker, and a curing target sequence;
- making cells of choice electrocompetent, wherein the cells of choice comprise an engine vector and the engine vector comprises a curing gRNA under the control of a second inducible promoter, a nuclease under the control of a third inducible promoter;
- and a second selectable marker;
- transforming the cells of choice with the first set of editing vectors to produce first transformed cells;
- selecting for the first transformed cells via the first and second selectable markers thereby selecting for first selected cells;
- inducing editing in the first selected cells by inducing the first and third inducible promoters thereby inducing transcription of the one or more editing gRNA and donor DNA pairs and the nuclease producing first edited cells;
- growing the first edited cells until the first edited cells reach a stationary phase of growth;
- curing the first set of editing vectors in the first edited cells by inducing the third and second inducible promoters thereby inducing transcription of the nuclease and curing gRNA which cuts the curing target sequence producing first cured cells;
- growing the first cured cells;
- rendering the first cured cells electrocompetent;
- and
- transforming the first cured cells with a second set of editing vectors to produce second transformed cells, wherein the second set of editing vectors comprises editing cassettes with one or more editing gRNA and donor DNA pairs operably linked and under the control of the first inducible promoter, a third selectable marker, and the curing target sequence.
10 more elements
A method for curing cells of choice during recursive nucleic acid-directed nuclease editing comprising:
- designing and synthesizing a first set of editing cassettes, wherein the first set of editing cassettes comprises one or more editing gRNA and donor DNA pairs wherein each editing gRNA and donor DNA pair is operably linked and under the control of a first inducible promoter;
- assembling the first set of editing cassettes into a vector backbone thereby forming a first set of editing vectors, wherein the vector backbone comprises a first selectable marker, a curing target sequence, and a curing gRNA under the control of a second inducible promoter;
- making cells of choice electrocompetent, wherein the cells of choice comprise an engine vector and the engine vector comprises a nuclease under the control of a third inducible promoter, and a second selectable marker;
- transforming the cells of choice with the first set of editing vectors to produce first transformed cells;
- selecting for the first transformed cells via the first and second selectable markers thereby selecting for first selected cells;
- inducing editing in the first selected cells by inducing the first and third inducible promoters thereby inducing transcription of the one or more editing gRNA and donor DNA pairs and nuclease producing first edited cells;
- growing the first edited cells until the first edited cells reach a stationary phase of growth;
- curing the first set of editing vectors in the first edited cells by inducing the third and second inducible promoters thereby inducing transcription of the nuclease and curing gRNA which cuts the curing target sequence producing first cured cells;
- growing the first cured cells;
- rendering the first cured cells electrocompetent;
- and
- transforming the first cured cells with a second set of editing vectors to produce second transformed cells, wherein the second set of editing vectors comprises editing cassettes with one or more editing gRNA and donor DNA pairs operably linked and under the control of the first inducible promoter, a third selectable marker, the curing target sequence, and the curing gRNA under the control of the second inducible promoter.
9 more elements
Dependencies are inferred from the claim text.