Multi-color droplet digital PCR assay enables allele-specific quantification of heterogeneous genome editing outcomes

Precise characterization of genome editing outcomes remains a major challenge because edited cell populations contain diverse alleles generated by homology-directed repair, non-homologous end joining (NHEJ), or base editing. While next-generation sequencing enables comprehensive analysis, its routine use is constrained by cost and turnaround time. Here, we developed a multi-color droplet digital PCR (ddPCR) assay that exploits six-color fluorescence detection to quantitatively distinguish multiple edited alleles within a single reaction. Using CRISPR-Cas9 and base editing model systems, we designed sequence-specific probe sets that distinguished recurrent NHEJ alleles generated by CRISPR-Cas9 editing as well as target and bystander alleles generated by base editing. The assay quantitatively resolved individual editing outcomes that could not be distinguished by conventional Sanger sequencing. Together, these results establish multi-color ddPCR as a rapid, scalable, and sequence-specific approach for quantification of genome editing outcomes across multiple editing modalities.