Joint single-cell profiling of Cas9 edits and transcriptomes reveals on- and off-target effects on gene expression (rhAmpSeq)
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: RhAmpseq was performed on two sets of eight genomic DNA samples. The first sample of each set was a control sample of unedited (electroporation only) K562 cells. The subsequent seven test samples were K562 cells edited with CRISPR-Cas9 and T7 promoter, using seven guides targeting chromatin remodeler genes <i>ARID1A</i>, <i>SMARCA4</i>, <i>CHD3</i>, and <i>CHD4</i>, one guide per sample. These eight samples were processed into rhAmpSeq libraries using one of two primer sets: either a multiplex panel of 52 primer pairs targeting Superb-seq detected and in silico predicted edit sites, or a singleplex primer pair targeting <i>ADSS1</i>. Sixteen total libraries, one replicate per sample and primer set.