Methods for measuring and improving CRISPR reagent function
Inventors
Kryukov, Gregory V. • Schlabach, Michael R. • Merkin, Jason J.
Assignees
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Publication Number
US-11946163-B2
Publication Date
2024-04-02
Expiration Date
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.
Core Innovation
The invention relates to a high-throughput CRISPR-sensor (CRISPR-SENSR™) platform that evaluates and optimizes gRNA function using sensor constructs. The sensor constructs contain each gRNA and a corresponding target sequence, including PAM/genomic context, and the platform introduces the sensor constructs into Cas9-expressing cells.
The platform uses amplification and sequencing of the sensor region to determine a pattern of edits. The sensor region sequence is used to detect genetic alterations, including insertions and deletions and specific base changes, including repair-template insertion, and the platform can also include transcription changes as part of the readout.
Based on the detected genetic alterations in the target sequence or an adjoining sequence, the platform provides determination of guide outcomes and supports distinguishing good versus bad guides using out-of-sample predictive modeling. The system further supports optimization of high-fidelity gRNAs and assembly of optimized gRNAs into genome-wide or subgenome-wide libraries, including disease- or gene-class targeted sets.
Documented results indicate that CRISPR-SENSR™ editing profiles match endogenous loci, enable improved dropout/enrichment and signal-to-noise by filtering edited reads, and support analysis of PAM/gRNA mismatch tolerance limits. The platform also supports predictive modeling of gRNA performance based on sequence features and demonstrates cutting efficiency and specificity across human cell lines.
Claims Coverage
The document explicitly provides one independent claim directed to detecting SNPs using an optimized high-fidelity gRNA library in Cas9-expressing cells, with amplification and sequencing of target regions and SNP determination from detected genetic alterations. The dependent claims further add core inventive limitations by specifying PAM definition, Cas9 variant choice, inducible control of Cas9 expression, and the genome-wide versus sub-genome-wide scope of the optimized gRNA library. Identified independent claim: clm-00001.
Optimized high-fidelity gRNA library in Cas9-expressing cells for target-region SNP readout
Introducing an optimized gRNA library comprised of high-fidelity gRNAs into a population of cells that have been modified to express Cas9 protein, culturing under conditions permitting expression of the gRNAs and the Cas9 protein, amplifying gRNA target sequences by PCR to obtain a plurality of target amplicons, sequencing the plurality of target amplicons, detecting genetic alterations in the target sequence, and determining the presence of one or more SNPs based on presence of genetic alterations in the target sequence or in an adjoining sequence.
Adjoining sequence defined as PAM for SNP determination
The adjoining sequence used for SNP determination is a protospacer adjacent motif (PAM).
Use of dCas9 protein
The Cas9 protein used is a dCas9 protein.
Use of Cas9 nickase mutant protein
The Cas9 protein used is a Cas9 nickase mutant protein.
Inducible gene element controlled Cas9 expression
Cas9 protein is encoded by a nucleic acid whose expression is controlled by an inducible gene element.
Genome-wide or sub-genome-wide optimized gRNA library scope
The optimized gRNA library is either genome-wide or sub-genome-wide.
Across the independent claim and its dependents, SNP detection is tied to introducing a high-fidelity optimized gRNA library into Cas9-expressing cells, PCR-amplifying and sequencing gRNA target regions, and determining SNP presence from detected genetic alterations in the target or adjoining sequence, with further limitations including use of PAM as the adjoining sequence, selecting dCas9 or Cas9 nickase mutant variants, requiring inducible regulation of Cas9 expression, and defining the gRNA library scope as genome-wide or sub-genome-wide.
Stated Advantages
Improved dropout/enrichment and signal-to-noise by filtering edited reads.
CRISPR-SENSR™ editing profiles match endogenous loci.
Supports out-of-sample predictive modeling of “good” versus “bad” guides using sequence features.
Documented Applications
Detecting the presence of one or more single nucleotide polymorphisms (SNPs) by amplifying and sequencing gRNA target sequences and determining SNP presence based on genetic alterations in the target sequence or an adjoining sequence.
Evaluating and optimizing gRNA function using a CRISPR-sensor (CRISPR-SENSR™) platform that determines edit patterns from sequencing outcomes.
Use of optimized gRNAs assembled into genome-wide or sub-genome-wide libraries, including disease- or gene-class targeted sets, for SNP detection and mutant-specific cutting.
Support out-of-sample predictive modeling of “good” versus “bad” guides and analysis of PAM/gRNA mismatch tolerance limits.
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