bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.25.761665

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

Launspach, M.·Raab, L. O.·Glaser, V.·Nitulescu, A.-M.·Beez, S.·Stein, M.·Hartmann, L. M.·Afzal, S.·Macos, J.·Quilalang, C. J.·Chen, J. J.·Nguyen, D. N.·Wilson, R. C.·Deubzer, H. E.·Wagner, D. L.·Kuenkele, A.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Launspach, M., Raab, L. O., Glaser, V., Nitulescu, A.-M., Beez, S., Stein, M., Hartmann, L. M., Afzal, S., Macos, J., Quilalang, C. J., Chen, J. J., Nguyen, D. N., Wilson, R. C., Deubzer, H. E., Wagner, D. L., Kuenkele, A.. 2026-10-27. CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in. https://doi.org/10.64898/2026.10.25.761665

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Sex-based differences in fibrosis markers in human abdominal aortic aneurysms

Background: Abdominal aortic aneurysms (AAA) are a complex degenerative vascular disease. The role of fibrosis in AAA progression is unclear. Our study evaluated the impact of fibroblast activation protein (FAP), collagen, and vimentin on AAA fibrosis and progression. We hypothesized that FAP may influence fibrosis and disease severity in AAA, with potential sex-specific differences. Study Design: Multi-centered transcriptomic analysis of aortic samples from 15 human AAA and 5 non-AAA was performed to evaluate cell-specific fibrosis gene expression profiles. Histopathological and molecular analysis of 26 male and female AAA samples (mAAA and fAAA) and 29 male and female normal abdominal aortas (mNAA and fNAA) was performed to assess whole-tissue fibrosis markers and signaling. Results: Single-cell RNA sequencing (scRNA-seq) analysis identified eight major cell clusters with distinct sex-based distributions. Although vascular smooth muscle cells (VSMCs) and fibroblast clusters had reduced counts in both male and female AAAs, they more highly expressed FAP and COL1A1 genes, particularly in females. Whole-tissue analysis demonstrated elevated extracellular matrix deposits with markedly increased collagen type 1 in both mAAA and fAAA. Alpha-smooth muscle actin (-SMA)-positive VSMCs were also reduced in mAAA and fAAA, but FAP was notably elevated, and robustly in fAAA. FAP-positive fibroblasts in fAAA exhibited increased expression of fibrosis markers, suggesting a unique pro-fibrotic phenotype. Conclusion: Our study reveals a sex-based difference in AAA tissue, with elevated fibrosis markers and increased FAP levels in females, suggesting its significant role in disease progression. These findings underscore the potential of FAP as a unique tissue biomarker for AAAs and warrants further investigation into novel targeted therapies that aim to stabilize AAAs, particularly in female patients.

molecular biology↗
bioRxiv2026

Endonuclease G governs an antiviral RNA surveillance system in budding yeast

Endonuclease G family DNA/RNA nucleases localize to the mitochondrial intermembrane space and prevent escape of mitochondrial DNA to the cytosol or nucleus. While extramitochondrial Endonuclease G also targets nuclear DNA during cell death, RNA regulatory roles of this family remain enigmatic. Here we discover an RNA surveillance system in Saccharomyces cerevisiae governed by Nuc1, the yeast Endonuclease G homolog. This system limits replication of L-A, an RNA mycovirus that endemically infects this organism. Whereas L-A resides in the cytosol, we find that Nuc1's antiviral function requires its mitochondrial intermembrane space localization. To understand Nuc1's indirect antiviral mechanism we investigated its genetic interactions with REX1 and REX2, RNA exonucleases that control 3' end processing of tRNA and rRNA intermediates in the nucleus. We demonstrate that the nuc1{triangleup} antiviral defect is massively amplified by rex1{triangleup} rex2{triangleup} leading to L-A RNA levels that surpass mature rRNAs and overwhelm the cells. NUC1 concurrently controls a pool of mis-processed tRNA and rRNA species that accumulate in rex1{triangleup} rex2{triangleup} mutants. Linking these mis-processed RNAs with L-A control, we show they are significantly reduced in L-A infected cells, suggesting that L-A depletes them to enhance its replication. Our findings identify a previously unrecognized RNA surveillance function of Nuc1 that prevents aberrant rRNA and tRNA species from promoting replication of the L-A mycovirus.

molecular biology↗
bioRxiv2026

Feedback control via H3K9 methylation and siRNA biogenesis autonomously maintains RNAi heterochromatin through continuous low-level transcription

The logic of RNA-based silencing involves nascent RNA molecules that serve as scaffolds to recruit proteins that repress transcription. Despite transcription and silencing being antagonistic, RNA-based recruitment mechanisms have been widely adopted in organisms ranging from yeast to humans. In fission yeast (Schizosaccharomyces pombe), the nuclear RNAi pathway that establishes heterochromatin at pericentromeric repeats to silence transcription exemplifies this paradox. Current models propose that cells reconcile these opposing activities by confining pericentromeric transcription to S phase while maintaining silencing during the rest of the cell cycle. Here, using strand-specific single-molecule RNA FISH (smFISH), we detect forward and reverse pericentromeric transcripts with high sensitivity and single-cell resolution. We find that repeat-associated transcription is rare, heterogeneous and happens independently of cell-cycle control. Instead, transcript production is governed solely by feedback involving small RNA biogenesis and H3K9 methylation. By combining mathematical modeling with experiments, we show that low levels of H3K9me or siRNAs prevent heterochromatin establishment, whereas high levels of H3K9me cause stochastic switching. Our work reveals that the same RNA-based feedback architecture can sample distinct transcriptional regimes, with feedback strength determining how often cells produce transcripts without any external regulatory inputs.

molecular biology↗