Multi-color droplet digital PCR assay enables allele-specific quantification of heterogeneous genome editing outcomes
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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.
Motivation
Genome editing generates complex outcomes of desired edits, wild-type alleles, and heterogeneous undesired edits. Although next-generation sequencing (NGS) provides comprehensive characterization of these outcomes, routine analysis remains limited by cost, turnaround time, and analytical complexity. Existing droplet digital PCR (ddPCR) assays offer a rapid and quantitative alternative but have been unable to distinguish individual NHEJ-derived indel alleles because of the limited number of fluorescence channels available in conventional platforms. In addition, characterization of base editing outcomes is complicated by bystander edits that cannot be resolved by standard Sanger sequencing. We sought to develop a versatile multi-color ddPCR strategy capable of directly detecting and quantifying multiple editing outcomes in a single reaction, thereby providing a rapid, cost-effective alternative to sequencing-based approaches for both CRISPR-Cas9 and base editing applications.
Highlights
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Six-color ddPCR assays simultaneously quantify WT, HDR, and multiple sequence-defined NHEJ alleles in a single reaction.
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The limits of detection for individual genome editing outcomes are as low as 0.08–0.30%.
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Multi-color ddPCR assays combined with machine learning–based indel prediction can capture more than 60% of editing outcomes without prior sequencing.
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Multi-color ddPCR assays enable discrimination of target and bystander modifications by base editing that cannot be resolved by Sanger sequencing.
