Biology subjects

Beez, S.

Publications and source records attributed to Beez, S..

2 recordsLinked to original sources

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↗

bioRxiv2025

Personalized CRISPR Knock-In Cytokine Gene Therapy to Remodel the Tumor Microenvironment and Enhance CAR T Cell Therapy in Solid Tumors

The immunosuppressive tumor microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumors. To address this, we developed a novel gene therapeutic strategy that enables localized remodeling of the TME via tumor-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumor-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma--a pediatric solid tumor with a suppressive TME--as a model, we applied CancerPAM to sequencing data from cell lines and patients to identify optimal integration sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG). CRISPR-mediated CXCL10 knock-in into tumor cells significantly enhanced CAR T cell infiltration and antitumor efficacy both in vitro and in vivo. In vivo, CXCL10-expressing tumors showed significantly increased early CAR T cell infiltration and prolonged survival compared to controls. CancerPAM rankings correlated strongly with target-site specificity and knock-in efficiency, validating its predictive performance. Our findings establish CancerPAM as a powerful tool for safe and effective CRISPR-based interventions and provide a conceptual framework for integrating cytokine-driven TME remodeling with cellular immunotherapies. This personalized strategy holds promise for enhancing CAR T cells and other immunotherapies across immune-refractory solid tumors.

bioengineering↗