Cell-in-drop genotyping detects megabase-scale loss of heterozygosity as a quantitative marker for CRISPR-Cas9 on-target toxicity

Targeted DNA double strand breaks catalyzed by CRISPR-Cas9 have been demonstrated to cause megabase-scale segmental deletions and whole chromosome losses in a wide variety of mouse and human cell lines. This raises safety concerns for CRISPR-Cas9 based therapeutics and highlights the need for scalable methods to assess its on-target genotoxicity in clinical contexts. In this report, we present cell-in-drop genotyping, as a high throughput approach that combines single cell encapsulation with droplet-based PCR genotyping of endogenous polymorphisms to detect loss of heterozygosity (LOH) at a targeted chromosome without any genome manipulation. With this approach, we detected megabase scale LOH induced by Cas9 DSBs, mostly frequently by segmental loss. This approach also detected LOH from loss of a critical DNA repair factor, BLM helicase, and LOH induced by other genotoxic conditions, in particular, the chemotherapeutic drugs Paclitaxel and Mitomycin C, which were demonstrated to induce LOH by different mechanisms. A modified version of the assay also captured rare local short range LOH arising from DSB-induced interhomologue recombination. Because it requires no genome manipulation, cell-in-drop genotyping offers a practical, scalable tool for evaluating genotoxic risk of CRISPR-Cas9 and other agents in research and clinical development.