Instruments, modules, and methods for improved detection of edited sequences in live cells
First Claim
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1. A method for enriching edited cells during CRISPR editing comprising:
- transforming cells with one or more vectors comprising an inducible promoter driving transcription of a coding sequence for a CRISPR nuclease, and an inducible promoter driving transcription of a guide nucleic acid sequence and a DNA donor sequence;
diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a substrate;
growing the cells on the substrate for 2-200 doublings;
initiating editing by inducing the inducible promoters driving transcription of the guide nucleic acid and CRISPR nuclease;
growing the induced cells into colonies; and
selecting or pooling slow-growing colonies on the substrate, wherein cells from the slow-growing colonies are enriched for edited cells.
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Abstract
The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.
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20 Claims
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1. A method for enriching edited cells during CRISPR editing comprising:
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transforming cells with one or more vectors comprising an inducible promoter driving transcription of a coding sequence for a CRISPR nuclease, and an inducible promoter driving transcription of a guide nucleic acid sequence and a DNA donor sequence; diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a substrate; growing the cells on the substrate for 2-200 doublings; initiating editing by inducing the inducible promoters driving transcription of the guide nucleic acid and CRISPR nuclease; growing the induced cells into colonies; and selecting or pooling slow-growing colonies on the substrate, wherein cells from the slow-growing colonies are enriched for edited cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for enriching edited cells during CRISPR nuclease editing comprising:
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transforming cells with one or more vectors comprising a promoter driving transcription of a coding sequence for a CRISPR nuclease, and an inducible promoter driving transcription of a guide nucleic acid sequence and a DNA donor sequence; diluting the transformed cells to a cell concentration to substantially singulate the transformed cells on a substrate; growing the cells on the substrate for between 2 and 200 doublings; initiating editing by inducing the inducible promoter; and growing the cells to form colonies of terminal size. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
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Specification