Joint single-cell profiling of CRISPR-Cas9 edits and transcriptomes reveals widespread off-target events and their effects on gene expression

A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to measure Cas9 edit outcomes and their functional effects at single-cell resolution. Here we present Superb-seq, a new technology that leverages T7 in situ transcription and single-cell RNA sequencing to jointly measure on- and off-target Cas9 edits and their effects on gene expression. We performed Superb-seq on 10,000 K562 cells, targeting four chromatin remodeler genes with seven guide RNAs. Superb-seq identified 11,891 edit events in 6,230 edited cells at all seven on-target sites and at an additional 36 off-target sites. Although the seven guides were selected for high specificity, six of them caused off-target edits, ranging in frequency from 0.03% to 18.6% of cells. A notable off-target edit within the first intron of USP9X disrupted the expression of this gene and over 150 downstream genes. In summary, Cas9 off-targeting is pervasive due to a combination of rare and common edit events, occurs primarily within introns of off-target genes, and can exert widespread effects on gene expression. Superb-seq uses off-the-shelf kits, standard equipment, and requires no virus, which will enable genome-wide CRISPR screens in diverse cell types as well as functional characterization of clinically-relevant guides.