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US 11,078,483 B1Granted
Methods for measuring and improving CRISPR reagent function
AssigneeKSQ Therapeutics, Inc.
InventorsGregory V. Kryukov, Michael R. Schlabach, Jason J. Merkin
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Abstract
The invention describes a novel system for identifying optimized gRNAs for use in CRISPR/Cas9 genome editing platforms. The invention allows for the determination of specific gene alterations rendered by a particular gRNA, thereby permitting the generation of optimized gRNA libraries.
Claims22
A method of preparing an optimized guide RNA (gRNA) library comprising;
- (a) introducing a library of nucleic acid constructs into a population of cells modified to express a Cas9 protein, wherein each construct comprises a gRNA sequence and a sensor sequence, wherein the sensor sequence comprises the corresponding gRNA target sequence;
- (b) culturing said cells from step (a) under conditions permitting the expression of said gRNA and said Cas9 protein;
- (c) amplifying the nucleic acid constructs sequences by polymerase chain reaction (PCR) from the cells of step (b) to obtain a plurality of amplicons, wherein each amplicon comprises the gRNA sequence and the sensor sequence;
- (d) sequencing said plurality of amplicons;
- (e) determining a pattern of alterations in the sensor sequence in each of the amplicons;
- and
- (f) identifying a population of gRNAs that produce a desired pattern of alterations in the sensor sequence,
- wherein the population of cells is modified with a nucleic acid encoding the Cas9 protein, and
- wherein the nucleic acid encoding the Cas9 protein mRNA is introduced into the population of cells by electroporation.
6 more elements
Dependencies are inferred from the claim text.