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

Cell-in-drop genotyping detects megabase-scale loss of heterozygosity after CRISPR-Cas9–induced double strand breaks, most often as segmental loss, providing a quantitative on-target toxicity marker.

• Why it matters: Safety concerns for CRISPR-Cas9 therapeutics require scalable ways to measure on-target genotoxicity in clinically relevant settings, including large chromosomal damage.
• What they did: The method encapsulates single cells and performs droplet-based PCR genotyping of endogenous polymorphisms to quantify LOH at a targeted chromosome without genome manipulation. It also captures rare local short-range LOH from DSB-induced interhomologue recombination.
• The result: The assay detects megabase-scale LOH from Cas9 DSBs, from loss of the DNA repair factor BLM helicase, and from Paclitaxel and Mitomycin C, enabling practical evaluation of genotoxic risk in development.

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