Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (RNA-seq)

A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to characterize editing outcomes at single-cell resolution. We present "Superb-seq", a new scRNA-seq method that measures on-target and off-target genome edits and associated transcriptomes. In contrast to Perturb-seq that captures single-cell guide information, Superb-seq directly captures single-cell edit profiles by T7 in situ transcription. We demonstrate and validate this method through two Superb-seq experiments in 30,000 cells of three cell types, amplicon sequencing (rhAmpSeq), and bulk RNA-seq. Superb-seq uses off-the-shelf kits, standard laboratory equipment, and requires no virus, which will improve CRISPR screens in diverse tissues and functional characterization of CRISPR therapeutics. Overall design: RNA-seq was performed on two sets of three human nuclei samples, one set per target site and cell type pair (CTLA4 in Jurkat, B2M in K562). For each set, first was a control sample with mock in situ transcription (no T7 RNA polymerase). Second was another control sample with CRISPR-Cas9 RNP only (no T7 promoter) and in situ transcription. Third was a test sample with both CRISPR-Cas9 RNP and T7 promoter, and in situ transcription. Two technical replicate libraries per sample.