High-throughput evaluation of in vitro CRISPR activities enables optimized large-scale multiplex enrichment of rare variants

Previous high-throughput evaluations of CRISPR activities for a large number of target and guide RNA sequences were based on measuring indel frequencies rather than cleavage efficiencies. Here we developed two high-throughput in vitro methods, Cut-seq1 and Cut-seq2, to evaluate Cas9 cleavage efficiency for tens of thousands or even hundreds of thousands of guide RNA-target pairs. These methods revealed low correlations between in vitro cleavage efficiencies and indel frequencies in cells, yet high concordances in protospacer adjacent motif compatibility. Using the resulting large datasets of in vitro cleavage efficiencies, we developed DeepCut, deep-learning models that can identify optimized sgRNAs that would selectively cleave specific sequences, even in the presence of similar noise sequences. Using these optimized sgRNAs, we developed a method, CLOVE-seq (Cleavage for Large-scale Optimized Variant Enrichment sequencing), to enrich rare variants in a multiplexed manner by Cas9-mediated specific cleavage of noise or rare variant sequences. Our methods can enhance the understanding of CRISPR nuclease activities and could be utilized to detect a large number of rare variants in various biomedical contexts.