Key result
ddXR accurately quantifies large DNA excisions and inversions, revealing unexpectedly high rates up to 170 kb.
Why the study?
Understanding factors that favor efficient DNA excision by paired CRISPR-Cas nucleases is limited by the lack of a rapid, accurate measurement method free of amplification bias.
The ddXR method provides a rapid, accurate, and amplification-bias-free approach to quantify large DNA excisions and inversions, facilitating disease modeling and therapeutic gene editing.
Enables rapid preclinical gene editing assessment in models; leaves open translation to human therapeutics.
The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR ( d roplet d igital PCR e X cision R eporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.
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Watry et al. (2020) studied Gene editing. ddXR (droplet digital PCR eXcision Reporter) vs. Traditional PCR and clone genotyping was evaluated on Detection and quantification of DNA excisions and inversions. The ddXR method enables rapid, accurate, and length-independent quantification of large DNA excisions and inversions, revealing unexpectedly high rates of large excisions up to 170 kb.
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