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 (<i>CTLA4</i> in Jurkat, <i>B2M</i> 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.