Biology subjects

Kuehn, R.

Publications and source records attributed to Kuehn, R..

13 recordsLinked to original sources

bioRxiv2026

Efficient CRISPR/Cas9-mediated homology independent sequence replacement in vivo and non-dividing primary cells

Precise sequence replacement in non-dividing cells remains a major challenge for genome editing. Here we show that REPLACE (Rational end-joining protocol delivering a targeted sequence exchange), a homology-independent CRISPR/Cas9-based replacement strategy, enables exon- and gene-scale substitution in primary cells, in vivo tissues and post-mitotic human cardiomyocytes. REPLACE uses two guide RNAs to excise a defined genomic region and inserts a donor lacking homology arms through non-homologous end joining (NHEJ). In primary mouse hepatocytes, REPLACE mediated exon replacement in 35% of all cells. In adult mouse liver, editing efficiency could be increased to [~]20% when Cas9-sgRNA ribonucleoproteins were delivered via engineered virus-like particles (eVLPs) together with an adeno-associated virus (AAV) donor. REPLACE also supported large-segment replacement, enabling one-step exchange of a [~]27-kb mouse Ace2 interval with the human ACE2 coding region in zygotes, followed by germline transmission and tissue-specific expression. Finally, we applied REPLACE to a disease-relevant mutation that is not readily addressable by base editing and was poorly corrected by prime editing in post-mitotic cardiomyocytes. At the LMNA locus, REPLACE corrected the K117fs frameshift mutation in patient-derived post-mitotic cardiomyocytes with precise exon replacement and restored Lamin A/C protein expression and nuclear lamina localization. These findings establish REPLACE as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable.

genomics↗

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↗

bioRxiv2026

Novel mouse reporter models for the detection of genome editing events in vivo

With the expansion of therapeutic gene editing technology, small animal models provide essential platforms to evaluate the function of these new approaches in vivo. As part of the Somatic Cell Genome Editing (SCGE) Consortium, we developed next-generation murine reporters that overcome current model limitations and broaden detectable in vivo editing outcomes. These include two mouse models built on the "traffic light" reporter concept. This system enables fluorescent detection of both gene repair (green) and CRISPR-generated indels (red) events following editing by a single guide and either dsDNA or single-stranded oligonucleotide donor. We also generated a third reporter model that efficiently detects A-base editor activity. Reporters were validated in cultured embryos, via germline editing, and through activation in vivo by AAV transduction or direct ribonucleoprotein delivery. Together, these new models provide a valuable resource for improved detection of genome editing events in vivo.

genomics↗

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↗

bioRxiv2024

Gene-edited primary muscle stem cells rescue dysferlin-deficient muscular dystrophy

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulated the patients phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells were able to regenerate muscle and rescued dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.

genetics↗

bioRxiv2023

Mutant Huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

Expansion of the glutamine tract (poly-Q) in the protein Huntingtin (HTT) causes the neurodegenerative disorder Huntingtons disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. 70Q introduction caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics and elevated resting energy expenditure. Elimination of the poly-Q tract of HTT normalized CHCHD2 expression and mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early intervention target for HD.

neuroscience↗

bioRxiv2021

Susceptibility to diet induced obesity at thermoneutral conditions is independent of UCP1

ObjectiveActivation of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) upon cold stimulation leads to substantial increase in energy expenditure to defend body temperature. Increases in energy expenditure after a high caloric food intake, termed diet-induced thermogenesis, are also attributed to BAT. These properties render BAT a potential target to combat diet-induced obesity. However, studies investigating the role of UCP1 to protect against diet-induced obesity are controversial and rely on the phenotyping of a single constitutive UCP1-knockout model. To address this issue, we generated a novel UCP1-knockout model by Cre-mediated deletion of Exon 2 in the UCP1 gene. We studied the effect of constitutive UCP1 knockout on metabolism and the development of diet-induced obesity. MethodsUCP1 knockout and wildtype mice were housed at 30{degrees}C and fed a control diet for 4-weeks followed by 8-weeks of high-fat diet. Body weight and food intake were monitored continuously over the course of the study and indirect calorimetry was used to determine energy expenditure during both feeding periods. ResultsBased on Western blot analysis, thermal imaging and noradrenaline test, we confirmed the lack of functional UCP1 in knockout mice. However, body weight gain, food intake and energy expenditure were not affected by deletion of UCP1 gene function during both feeding periods. ConclusionConclusively, we show that UCP1 does not protect against diet-induced obesity at thermoneutrality. Further we introduce a novel UCP1-KO mouse enabling the generation of conditional UCP1-knockout mice to scrutinize the contribution of UCP1 to energy metabolism in different cell types or life stages.

physiology↗

bioRxiv2021

In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation

Vertebrate genomes organize into topologically associating domains (TADs), delimited by boundaries that insulate regulatory elements from non-target genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CTCF boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding sites (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors like CBS number and orientation. Combined deletions demonstrated that CBS cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by non-convergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

developmental biology↗

bioRxiv2020

A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

Precision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method Replace editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

genetics↗

bioRxiv2019

SURF1 mutations causative of Leigh syndrome impair human neurogenesis

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

neuroscience↗

bioRxiv2017

Targeted re-sequencing of coding DNA sequences for SNP discovery in non-model species

Hybridization capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. A cross-species approach makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for non-model organisms, for which often no a priori knowledge exists regarding these loci. Here, we have adopted cross-species capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a non-model organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). In this manner, we were able to survey intraspecific variation at hundreds of nuclear loci across the European range of L. lynx. A large set of bi-allelic candidate SNPs was then tested in a high throughput SNP-genotyping platform (Fluidigm), which we reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with additional samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (incl. the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.

genetics↗

bioRxiv2016

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g., by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.

genetics↗

bioRxiv2016

Taming the Late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA

Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into nine clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent gene flow events between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly endangered species.

evolutionary biology↗