Methods for measuring and improving crispr reagent function
AssigneeKSQ Therapeutics, Inc.
InventorsGregory V. Kryukov, Michael R. Schlabach, Jason J. Merkin
Life of this patent
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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.
Claims27
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.
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An optimized guide RNA (gRNA) library prepared by:
- (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;
- (b) culturing said cells from step (a) under conditions permitting the expression of said gRNA and said Cas9 protein;
- (c) amplifying the nucleic acid construct sequences by polymerase chain reaction (PCR) from the cells of step (b) to obtain a plurality of amplicons, wherein each amplicon comprises a gRNA nucleic acid sequence and a sensor sequence;
- (d) sequencing said plurality of amplicons;
- (e) determining a pattern of alterations in the sensor sequence in each of said amplicons;
- and
- (f) identifying a population of optimized gRNAs that produce a desired pattern of alterations in said sensor sequence;
- and
- (g) preparing a library of said optimized gRNAs.
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A method of detecting the presence of one or more single nucleotide polymorphisms (SNPs) comprising:
- (a) introducing an optimized gRNA library comprised of high-fidelity gRNAs into a population of cells that have been modified to express Cas9;
- (b) culturing said cells from step (a) under conditions permitting the expression of said gRNA and said Cas9 protein;
- (c) amplifying gRNA target sequences by polymerase chain reaction (PCR) from the cells of step (b) to obtain a plurality of target amplicons;
- (d) sequencing said plurality of target amplicons;
- (e) detecting the presence of genetic alterations within the target sequence;
- and
- (f) determining the presence of one or more SNPs based on the presence of genetic alterations within the target sequence, or in adjoining sequences such as the protospacer adjacent motif (PAM).
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Dependencies are inferred from the claim text.