Analysis of Genome Editing Alterations and Functional Outcomes in CRISPR-Engineered Cells

CRISPR-Cas genome-editing holds great therapeutic promise but is limited by off-target activity (OTA), which can cause unintended mutations and structural variations. Most studies assess OTA at the bulk level, missing single-cell resolution and functional consequences of editing. To address this, we applied the Tapestri platform—single-cell DNA sequencing integrated with multi-omics profiling—to analyze human primary T-cells edited with three gRNAs from a first-in-human CRISPR trial (targeting PDCD1, TRAC, TRBC). We sequenced 111 putative off-target sites across thousands of cells, comparing results with rhAmpSeq bulk sequencing. While overall editing rates were similar, single-cell data revealed diverse zygosity and unique editing profiles per cell, including novel translocations. A 12-day longitudinal study linked bi-allelic inDels with reduced TCRa/b and CD3 protein expression. Our findings underscore the value of single-cell analysis in identifying editing heterogeneity and linking genotypes to phenotypes, offering a powerful framework for quality control in genome-engineered cell therapies.