Raab, L. O. | Search bioRxiv

bioRxiv2026

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

Accurate characterization of CRISPR knock-in requires distinguishing site-specific integration from total donor abundance and donor-derived background while measuring editing at the target site. Here, we present CRISPR-dPCR, a modular multiplex digital PCR framework combining a locked nucleic acid-enhanced cut-site assay with junction-based detection and two orthogonal short-amplicon strategies: Poisson linkage and homology-arm subtraction. We evaluated assay modules across genomic loci, neuroblastoma models and primary human T cells, with selected assays compared on two dPCR platforms. In genotype-defined clones and mixtures with low residual donor, homology-arm subtraction most closely recovered expected knock-in abundance. Linkage provided highly specific evidence of target-cargo association without junction-spanning amplification, whereas junction assays supplied boundary-specific confirmation but were more sensitive to amplicon length and multiplexing. Five-channel panels integrated cut-site editing, donor abundance, copy-number normalization and site-specific knock-in in a single reaction. Residual donor DNA limited quantitative interpretation, particularly in early post-editing samples. Modular extensions quantified defined unintended outcomes, including chromosomal translocations. CRISPR-dPCR integrates complementary measurements into a scalable analytical framework for genome-editing research, cell and gene therapy product development and applications in longitudinal clinical monitoring of targeted integration and transgene persistence.

molecular biology↗

bioRxiv2026

A Non-Viral CRISPR/Cas9 HDR Platform for Stable Engineering of Solid Tumor Models.

Virus-free genome engineering provides a flexible alternative to viral vectors for generating genetically modified cell models. Here, we establish an integrated biosafety level 1-compatible CRISPR/Cas9 homology-directed repair (HDR) workflow for stable transgene knock-in in neuroblastoma cell lines using non-viral delivery approaches. We systematically evaluated donor cassette architecture and delivery conditions across electroporation-based Cas9 ribonucleoprotein (RNP) delivery and lipid nanoparticle (LNP)-mediated co-delivery of Cas9 mRNA, sgRNA, and donor DNA. Modular AAVS1-targeting donor constructs identified a compact EF1(s)-Donor-Q8-Tag-sPA cassette that consistently yielded the strongest HDR-associated knock-in readouts, achieving up to 60% stable reporter-positive cells following electroporation without HDR enhancers. While LNP-mediated delivery enabled efficient CRISPR cargo co-delivery and generation of genetically modified tumor cell populations, knock-in efficiencies remained lower than those observed with electroporation. Subsequent enrichment approaches enabled generation of highly pure edited cell populations following both delivery strategies. Functional validation demonstrated stable transgene expression in vitro, including in three-dimensional bioprinted tumor models, and in vivo in xenograft mice without impairing tumor growth or viability. Together, these findings establish a practical non-viral HDR platform for stable engineering of solid tumor models and provide a framework for further optimization of genome editing workflows across distinct delivery modalities. Key findings- We establish a complete, virus-free CRISPR/Cas9 HDR workflow that reliably enables stable knock-in in solid tumor cell lines, demonstrated here in two neuroblastoma models under biosafety level 1 conditions. - We establish and evaluate LNP-mediated co-delivery of Cas9 mRNA, gRNA, and donor DNA for non-viral HDR knock-in in solid tumor models, revealing delivery modality-specific differences in editing efficiency, toxicity, and expression dynamics. - By systematically varying donor architectures, we identify a compact HDR template - combining a shortened custom EF1 promoter, the minimal Q8 surface reporter, and a synthetic polyadenylation signal (sPA) - that markedly improves knock-in efficiency in solid tumor cell lines, outperforming conventional cassettes. - Virus-free edited tumor cells generated using this workflow retain stable transgene expression and functional fitness in 3D bioprinted tumor constructs and xenograft mouse models, directly linking in vitro knock-in optimization to in vivo relevance. - The resulting biosafety level 1 compatible, end-to-end pipeline - integrating donor design, digital PCR-based quantification of precise integration, and enrichment strategies-offers a practical and transferable platform for engineering transgenic solid tumor models without viral vectors.

bioengineering↗