Compartmentalized CRISPR Reactions (CCR) for High‐Throughput Screening of Guide RNA Potency and Specificity

Abstract

AbstractCRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target‐specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably in a spacer sequence‐dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effector's activity and/or potential for off‐target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, the study presents compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off‐target combinations simultaneously in vitro for both CRISPR effector activity and specificity by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water‐in‐oil microemulsions. With CCR, large numbers of the candidate gRNAs (output by computational design tools) can be immediately validated in parallel, and the study shows that CCR can be used to screen hundreds of thousands of extended gRNA (x‐gRNAs) variants that can completely block cleavage at off‐target sequences while maintaining high levels of on‐target activity. It is expected that CCR can help to streamline the gRNA generation and validation processes for applications in biological and biomedical research.