Joint single-cell profiling of Cas9 edits and transcriptomes determines on- and off-target regulatory effects

A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to measure Cas9 edit outcomes at single-cell resolution. Here we present Superb-seq, a new technology for joint measurement of on-target and off-target Cas9 edits and transcriptomes by single-cell RNA sequencing. In contrast to Perturb-seq methods that read each cell’s guide RNA, Superb-seq directly reads Cas9 edits by leveraging T7 in situ transcription. We performed Superb-seq on 9,500 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 36 off-target sites. One notable off-target edit fell within the first intron of the deubiquitinase gene USP9X, which decreased its expression and perturbed the expression of downstream genes. Superb-seq uses off-the-shelf kits, standard equipment, and requires no virus, which will enable CRISPR screens in virus-intolerant cell types and functional characterization of off-target events. To demonstrate that Cas9 genome edits can be identified through T7 RNA, we performed T7 in situ transcription and bulk RNA-seq on human nuclei containing T7 promoter-labeled edits. To demonstrate single-cell edit capture and gene expression association, we performed Superb-seq on K562 cells edited at four chromatin remodeler genes (ARID1A, SMARCA4, CHD3, CHD4) with seven guide RNAs, and analyzed two single-cell libraries of 500 and 10,000 cells.