Comprehensive On-and Off-target Confirmation Using Integrated rhAmpSeq and Targeted DNA Sequencing Single-Cell Technology

Advances in genome editing have enabled precise correction of mutations and engineering of cells with therapeutic functions. A key factor in successful CRISPR editing is assay specificity, where guide RNAs (gRNAs) direct accurate edits with minimal off-target effects. Amplicon-based targeted resequencing is the gold standard for validating edits due to its high sensitivity, multiplexing capability, and cost-effectiveness. Innovations like rhAmpSeq improve bulk validation by reducing background noise in multiplexed PCR through optimized primer chemistry. Given that cells are the functional units of gene editing, it is crucial to assess edit outcomes and genotoxicity at the single-cell level. Droplet-based single-cell DNA resequencing offers insights into zygosity, edit co-occurrence, and clonality. In this study, we integrated rhAmpSeq with droplet-based single-cell assays (Tapestri platform) to create a flexible workflow for single-cell resolution validation. This approach—encompassing rhAmpSeq panel design, single-cell chemistry, and the Tapestri Genome Editing (GE) Pipeline—enables detailed, accurate analysis of gene editing outcomes and potential malignant events.