CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in

CRISPR-dPCR most closely recovered expected CRISPR knock-in abundance in genotype-defined clones and low-residual-donor mixtures using homology-arm subtraction, while residual donor limited early post-editing quantification.

• Why it matters: Accurate knock-in characterization must separate site-specific integration from total donor abundance and donor-derived background while also measuring target-site editing.
• What they did: The modular multiplex framework combines a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction, evaluated across genomic loci, neuroblastoma models, and primary human T cells.
• The result: Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization, and site-specific knock-in in one reaction, enabling boundary-specific confirmation and scalable monitoring, with modular extensions quantifying unintended outcomes including chromosomal translocations.