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US 11,597,923 B2Granted
CRISPR editing to embed nucleic acid landing pads into genomes of live cells
AssigneeInscripta, Inc.
InventorDaniel Held
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Abstract
The present disclosure relates to compositions, methods, modules and automated integrated instrumentation for multiplex delivery of “landing pad” edits into the genomes of a population of live cells. The landing pads then may be leveraged to insert very large DNA sequences into the genomes of the population of live cells.
Claims31
A method for multiplex insertion of large DNA payloads into a population of cells comprising the steps of:
- designing and synthesizing a library of editing cassettes comprising landing pads, wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking each landing pad;
- inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors;
- transforming the population of cells with the library of editing vectors;
- allowing editing to take place in the population of cells to produce edited cells;
- transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for an integrase under the control of an inducible promoter;
- and
- inducing expression of the integrase to insert the large DNA payloads into the landing pads.
4 more elements
A method for multiplex insertion of large DNA payloads into a population of cells comprising the steps of:
- obtaining a library of editing vectors comprising a library of editing cassettes comprising landing pads, wherein the landing pads comprise a recognition sequence for an integrase, and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking each landing pad;
- transforming the population of cells with the library of editing vectors;
- allowing editing to take place in the population of cells to produce edited cells;
- transforming the edited cells with (i) one or more vectors carrying one or more large DNA payloads and (ii) a coding sequence for the integrase;
- and
- allowing the integrase to insert the one or more large DNA payloads into the landing pads.
3 more elements
Dependencies are inferred from the claim text.