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

ABSTRACT: 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

SUBMITTER: Lorenzini MH 

PROVIDER: S-EPMC12407703 | biostudies-literature | 2025 Aug

REPOSITORIES: biostudies-literature