Biology subjects

Hartmann, L. M.

Publications and source records attributed to Hartmann, L. M..

5 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↗

bioRxiv2026

Horizontal transfer promotes allele segregation in multicopy plasmids

Plasmids reside within prokaryotic cells in multiple copies and can spread horizontally between hosts. Their multicopy nature enables intracellular allele diversity (heteroplasmy), and their segregation depends on the modes of plasmid replication and partition. Horizontal plasmid transfer has the potential to alter plasmid allele composition, however its impact on plasmid allele dynamics remains poorly understood. Here, we show that conjugative plasmid transfer accelerates the segregation of plasmid heteroplasmy by promoting the emergence of homoplasmic hosts. Using a quantitative experimental system to track plasmid allele dynamics in Acinetobacter baylyi under non-selective conditions, we followed the fate of a novel antibiotic resistance allele introduced into an ancestral donor population. While alleles in heteroplasmic donors segregated over time, conjugation produced almost only homoplasmic recipients whose allele composition closely mirrored that of the donor pool. Heteroplasmic recipients were rare and arose primarily from multiple plasmid transfer events prior to plasmid establishment. A mathematical model calibrated to the experimental setup predicts that conjugation accelerates plasmid allele segregation, with effects scaling with plasmid copy number and conjugation frequency. Our findings identify horizontal transfer as a previously unrecognized segregation pathway shaping the evolution of mobile genetic elements.

evolutionary biology↗

bioRxiv2025

Repurposing base editors for targeted knock-in and simultaneous knockouts to generate multiplex-edited allogeneic CAR T cells with minimal translocations

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed Base editor-mediated knock-in (BEKI), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC, CD3{zeta}, B2M, and CD3{varepsilon} loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/676172v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f5f6e5org.highwire.dtl.DTLVardef@246411org.highwire.dtl.DTLVardef@125a478org.highwire.dtl.DTLVardef@510278_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Versatile and efficient non-viral integration of large transgenes in human T cells via CRISPR knock-in and engineered integrases

Current gene transfer methods often lack the precision, versatility, or efficiency when integrating large transgenes, limiting the ability to engineer therapeutic T-cells with more complex payloads. Here, we report one-pot PASTA (Programmable and Site-specific Transgene Addition), a non-viral genome engineering strategy for large gene insertion that combines CRISPR-Cas-mediated homology-directed repair (HDR) and site-specific recombination via serine integrases. Using one-pot PASTA with the Bxb1 integrase, we demonstrate efficient integration of transgenes at multiple genomic loci relevant for T-cell engineering (e.g., TRAC, B2M, CD3E, CD3Z, GAPDH). For constructs > 8 kb, one-pot PASTA outperforms conventional HDR by 19-fold on average and prime-editing-assisted site-specific integrase gene editing (PASSIGE) by 5-fold. This enables the delivery of multi-cistronic cargo to generate dual-antigen targeting CAR T-cells with a safety-switch that overcome antigen escape in lymphoma models. Finally, one-pot PASTA can be further optimized with improved integrase enzymes, such as engineered variants of Pa01 or Bxb1, and plasmids with minimized backbones. In summary, one-pot PASTA represents a versatile and scalable platform for precise, non-viral gene insertion in T-cells.

synthetic biology↗

bioRxiv2019

Neural correlates of exceptional memory ability in SuperAgers: A multimodal approach using FDG-PET, PIB-PET, and MRI

Individuals at 80 years of age or above with exceptional memory are considered SuperAgers (SA). A multimodal brain analysis of SA may provide biomarkers of successful cognitive aging. Herein, a molecular (PET-FDG, PET-PIB), functional (fMRI) and structural analysis (MRI) of SA was conducted. Ten SA, ten age-matched older adults (C80) and ten cognitively normal middle-aged adults underwent cognitive testing and neuroimaging examinations. The relationship between cognitive scores and cingulate areas and hippocampus were examined. The SA group showed increased FDG SUVr in the left subgenual Anterior Cingulate Cortex (sACC, p<0.005) as compared to that in the C80 group. Amyloid deposition was similar between SA and C80 in the described regions or overall areas (p>0.05). The SA group also presented decreased connectivity between left sACC and posterior cingulate (p<0.005) as compared to that of C80 group. These results support the key role of ACC in SA, even in the presence of amyloid deposition. It also suggests that sACC can be used as a potential memory biomarker in older adults.\n\nAbbreviationsBCa - Bias corrected accelerated: SA - SuperAgers: C50 - Middle-aged controls: C80 - Age-matched controls

neuroscience↗