Biology subjects

Concordet, J.-P.

Publications and source records attributed to Concordet, J.-P..

17 recordsLinked to original sources

bioRxiv2026

Error-free and efficient prime editing in absence of maternal Polθ

Prime editing is a versatile genome-editing technology developed to make small edits while minimising the unwanted mutations associated with approaches based on double-strand breaks. In zebrafish embryos, however, prime editing is highly error-prone, typically yielding more unwanted mutations than precise edits. Here, we show that most unwanted mutations are deletions flanked by microhomologies and insertions templated from neighbouring sequences, both hallmarks of microhomology-mediated end-joining of double-strand breaks. These breaks are unlikely to result from direct cleavage of both genomic strands by the prime editor. Instead, we propose that the rapid cell divisions of early zebrafish embryos promote the conversion of nicks into double-strand breaks by replication forks. Loss of maternally deposited DNA polymerase {theta} (Pol{theta}), the core factor in microhomology-mediated end-joining, abolishes unwanted mutations and can increase edit rates above 50%, resulting in error-free and efficient prime editing. Our work identifies the source of unwanted prime-editing mutations in zebrafish and establishes a practical strategy to achieve high edit rates and near-perfect precision.

genetics↗

bioRxiv2025

AAV-delivered CRISPR-Cas9 elicits persistent retinal immune responses compared with transient responses to RNP

CRISPR-Cas9 is a powerful gene-editing tool with great potential for treating genetic diseases, including inherited retinal disorders. However, its bacterial origin can induce immune responses that may eliminate transduced cells, threatening editing efficiency. A deeper understanding of CRISPR-Cas9 immunogenicity is therefore needed. Previous studies have shown that systemic delivery via Cas9 induces an immune response, but the detailed inflammation and the impact of the vector remain unclear, especially in immune-privileged organs like the eye. In this study, we found that Cas9 delivered to the retina using adeno-associated virus (AAV) induced persistent inflammation, whereas delivery as naked ribonucleoprotein (RNP) complexes resulted in acute inflammation that faded three weeks post-injection. Inflammation was more severe in the rd10 mouse model of inherited retinal degeneration, which exhibits basal inflammation. These findings provide new insights into vector-dependent immune responses to Cas9 in the eye and highlight potential risks associated with its clinical application. TEASERUnderstanding immune reactions to CRISPR-Cas9 and linking these to their delivery methodology increases their safety.

bioengineering↗

bioRxiv2025

Stress-Induced Nuclear Import Governs Transcription Recovery after DNA Damage

Genotoxic stress inhibits transcription, generating RNA-DNA hybrid intermediates that promote toxic transcription-replication conflicts (TRCs). Here, we show that genotoxic stress triggers transient Importin-/{beta}1-dependent nuclear import of the mitochondrial exonuclease EXD2 to resolve these conflicts and maintain genome stability. Mechanistically, once imported into the nucleus, EXD2 is recruited to elongation-arrested RNA polymerase II (RNAPII) associated with RNA-DNA hybrids, and its exonuclease activity limits their accumulation, thereby suppressing TRCs. Mutation of a putative EXD2 nuclear localization signal phenocopies disruption of nuclear import, whereas forced nuclear targeting of EXD2 bypasses it. However, sustained nuclear localization of EXD2 leads to mitotic defects, underscoring the importance of its tight spatial regulation. Consistent with a direct role for EXD2 in RNA-DNA hybrid resolution, overexpression of the ribonuclease RNaseH1 compensates for its depletion following DNA damage. Together, our findings introduce the concept of a trafficking-dependent checkpoint that transiently licenses EXD2 nuclease activity in the nucleus in response to genotoxic stress to resolve RNA-DNA hybrid intermediates at stalled RNAPII complexes TeaserStress-triggered nuclear import of EXD2 resolves harmful RNA-DNA hybrids to protect genome stability after DNA damage.

molecular biology↗

bioRxiv2024

Retinal organoids mirror CRISPR/Cas9 gene editing efficiency observed in vivo

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include the presence of all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing optimization, particularly for specific genetic disorders. We assessed the potential of retinal organoids for optimizing CRISPR/Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant Retinitis Pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR/Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual AAV systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/630388v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@94478borg.highwire.dtl.DTLVardef@e50970org.highwire.dtl.DTLVardef@ce998corg.highwire.dtl.DTLVardef@1c2bbdf_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOCRetinal organoids can be used to mirror in vivo mouse retina to develop CRISPR therapeutics. Here, Pulman and colleagues show similar ranges of gene editing and delivery dynamics between the organoids and in vivo mouse retina, highlighting the organoids underexplored potential for evaluating gene editing therapies in retinal diseases.

bioengineering↗

bioRxiv2024

Safety and efficacy study of CRISPR/Cas9 treatment of sickle cell disease in clinically relevant conditions highlights disease-specific response

Reactivation of fetal hemoglobin (HbF) expression through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated disruption of regulatory elements involved in {gamma}-globin gene repression is a promising gene therapy strategy for the treatment of sickle cell disease (SCD). However, preclinical studies aimed at optimizing the genome editing process and evaluating the safety of the editing strategy are necessary to translate this approach to the clinics. This is particularly relevant in the context of SCD, a disease characterized by inflammation, which can affect hematopoietic stem and progenitor cells (HSPCs), the target cell population in gene therapy approaches for hematopoietic disorders. Here, we describe a genome editing strategy leading to therapeutically relevant reactivation of HbF expression by targeting the binding sites (BSs) for the leukemia/lymphoma related factor (LRF) transcriptional repressor in the HBG1 and HBG2 {gamma}-globin promoters. Electroporation of Cas9 ribonucleoprotein and single guide RNA (sgRNA) targeting the HBG promoters in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny differentiated in vitro and ex vivo after transplantation into immunodeficient mice. LRF BS disruption did not impair SCD and HD HSPC engraftment and differentiation, but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared to HD cells. Importantly, in HSPCs, we detected off-target activity and the intra- and inter-chromosomal rearrangements between on- and off-target sites, which were more pronounced in SCD samples (likely because of the higher overall editing efficiency), but did not impact the target gene expression. Off-target activity was observed in vitro and in vivo, thus indicating that it does not impair engraftment and differentiation of SCD and HD HSPCs. Finally, transcriptomic analyses showed that the genome editing procedure results in the upregulation of genes involved in DNA damage and inflammatory responses in both HD and SCD samples, although gene dysregulation was more evident in SCD HSPCs. Overall, this study provides evidences of feasibility, efficacy and safety for a genome editing strategy based on HbF reactivation and highlights the need of performing safety studies, when possible, in clinically relevant conditions, i.e., in patient-derived HSPCs.

bioengineering↗

bioRxiv2023

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR- Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Successful delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes has been reported in the retinal pigment epithelium in vivo but not into photoreceptors, the main target of retinal dystrophies. Here, we investigate the feasibility of direct RNP delivery to photoreceptors and RPE cells. We show that RNPs composed of Cas9 or adenine- base editor and guide RNA, without addition of any carrier compounds, induce gene editing in retinal cells at variable rates depending on the guide RNA efficiency and on the locus. But Cas9 RNP delivery at high concentrations leads to outer retinal toxicity indicating a need to improve delivery efficiency for future therapeutic use.

bioengineering↗

bioRxiv2023

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation

Beyond the observed alterations in cellular structure and mitochondria, the cellular mechanisms linking genetic mutations to the development of heart failure in patients affected by desmin defects remain unclear due, in part, to the lack of relevant human cardiomyocyte models. We investigated the role of mitochondria using cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K desmin mutation, that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cells were either cultured on an anisotropic surface to obtain elongated and aligned cardiomyocytes, or as spheroids to create a micro- tissue. When applicable, results were confirmed with heart biopsies from the family harboring DESE439K mutation. We show that mutant cardiomyocytes reproduce critical defects in mitochondrial architecture, respiratory capacity and metabolic activity as observed in patients heart tissue. To challenge the pathological mechanism, normal mitochondria were transferred inside the mutant cardiomyocytes. This treatment restored mitochondrial and contractile functions. This work demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens-up new potential therapeutic perspectives.

pathology↗

bioRxiv2023

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicals in vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily. To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model species Hypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species, H. exemplaris, Acutuncus antarcticus and Paramacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, named Tardigrade DNA damage Response protein 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

molecular biology↗

bioRxiv2023

Changes in social behaviour with alterations of MAPK3 and KCTD13/CUL3 pathways in two new outbred rat models for the 16p11.2 syndromes with autism spectrum disorders.

Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed convergent defects in social behaviour and only a few cognitive defects. Interestingly major pathways affecting MAPK3 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behaviour are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism, with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behaviour and understanding the brain mechanisms and specific brain regions that are key to controlling social behaviour.

animal behavior and cognition↗

bioRxiv2021

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo.

Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing the NRN PAM sequence, we could introduce point mutations for the first time in an animal model and achieved up to 100% efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we applied our method to create a new zebrafish model for melanoma predisposition based on the simultaneous editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

developmental biology↗

bioRxiv2021

A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

The contractile properties of adult myofibers are shaped by their Myosin heavy chain (MYH) isoform content. We identify by snATAC-seq a 42kb super-enhancer (SE) at the locus regrouping the fast Myh (fMyh) genes. By 4C-seq we show that active fMyh promoters interact with the SE by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the SE recapitulates the endogenous spatio-temporal expression of adult fMyh genes. In situ deletion of the SE by CRISPR/Cas9 editing demonstrates its major role in the control of associated fMyh genes, and deletion of two fMyh genes at the locus reveals an active competition of the promoters for the shared SE. Last, by disrupting the organization of fMyh, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

cell biology↗

bioRxiv2020

Precise base editing for the in vivo study of developmental signaling and human pathologies in zebrafish

While zebrafish is emerging as a new model system to study human diseases, an efficient methodology to generate precise point mutations at high efficiency is still lacking. Here we show that base editors can generate C-to-T point mutations with high efficiencies without other unwanted on-target mutations. In addition, we established a new editor variant recognizing an NAA PAM, expanding the base editing possibilities in zebrafish. Using these approaches, we first generated a base change in the ctnnb1 gene, mimicking oncogenic mutations of the human gene known to result in constitutive activation of endogenous Wnt signaling. Additionally, we precisely targeted several cancer-associated genes among which cbl. With this last target we created a new zebrafish dwarfism model. Together our findings expand the potential of zebrafish as a model system allowing new approaches for the endogenous modulation of cell signaling pathways and the generation of precise models of human genetic disease associated-mutations.

developmental biology↗

bioRxiv2019

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

cell biology↗

bioRxiv2019

Identification of the master sex determining gene in Northern pike (Esox lucius) reveals restricted sex chromosome differentiation

Teleost fishes, thanks to their rapid evolution of sex determination mechanisms, provide remarkable opportunities to study the formation of sex chromosomes and the mechanisms driving the birth of new master sex determining (MSD) genes. However, the evolutionary interplay between the sex chromosomes and the MSD genes they harbor is rather unexplored. We characterized a male-specific duplicate of the anti-Mullerian hormone (amh) as the MSD gene in Northern Pike (Esox lucius), using genomic and expression evidences as well as by loss-of-function and gain-of-function experiments. Using RAD-Sequencing from a family panel, we identified Linkage Group (LG) 24 as the sex chromosome and positioned the sex locus in its sub-telomeric region. Furthermore, we demonstrated that this MSD originated from an ancient duplication of the autosomal amh gene, which was subsequently translocated to LG24. Using sex-specific pooled genome sequencing and a new male genome sequence assembled using Nanopore long reads, we also characterized the differentiation of the X and Y chromosomes, revealing a small male-specific insertion containing the MSD gene and a limited region with reduced recombination. Our study depicts an unexpected level of limited differentiation within a pair of sex chromosomes harboring an old MSD gene in a wild population of teleost fish, highlights the pivotal role of genes from the amh pathway in sex determination, as well as the importance of gene duplication as a mechanism driving the turnover of sex chromosomes in this clade. Author SummaryIn stark contrast to mammals and birds, teleosts have predominantly homomorphic sex chromosomes and display a high diversity of sex determining genes. Yet, population level knowledge of both the sex chromosome and the master sex determining gene is only available for the Japanese medaka, a model species. Here we identified and provided functional proofs of an old duplicate of anti-Mullerian hormone (Amh), a member of the Tgf-{beta} family, as the male master sex determining gene in the Northern pike, Esox lucius. We found that this duplicate, named amhby (Y-chromosome-specific anti-Mullerian hormone paralog b), was translocated to the sub-telomeric region of the new sex chromosome, and now amhby shows strong sequence divergence as well as substantial expression pattern differences from its autosomal paralog, amha. We assembled a male genome sequence using Nanopore long reads and identified a restricted region of differentiation within the sex chromosome pair in a wild population. Our results provide insight on the conserved players in sex determination pathways, the mechanisms of sex chromosome turnover, and the diversity of levels of differentiation between homomorphic sex chromosomes in teleosts.

evolutionary biology↗

bioRxiv2018

HSF2 protects against proteotoxicity by maintaining cell-cell adhesion

Cellular ability to maintain proper protein homeostasis (proteostasis) is essential for survival upon protein-damaging conditions. Heat shock transcription factor 2 (HSF2) is one of the human HSFs activated in response to proteotoxic stress. HSF2 is dispensable for cell survival during acute heat stress, but its amount and DNA-binding activity increase under prolonged proteotoxic stress conditions, such as proteasome inhibition. Nevertheless, the specific role(s) of HSF2 and the global HSF2-dependent gene expression profile during sustained stress have remained elusive. We found that HSF2 is required for cell survival during prolonged proteotoxicity, as shown by treating wild-type and HSF2-deficient human osteosarcoma U2OS cells with the proteasome inhibitor Bortezomib. Strikingly, our RNA-seq analyses revealed that HSF2 disruption leads to marked downregulation of cadherin superfamily genes and subsequent functional impairment of cadherin-mediated cell-cell adhesion. We propose HSF2 as a key regulator of genes belonging to the cadherin superfamily. We also demonstrate that HSF2-dependent downregulation of cadherin-mediated cell-cell adhesion predisposes U2OS cells to Bortezomib-induced proteotoxic stress. In conclusion, we show that by maintaining cell-cell adhesion HSF2 is essential for cell survival and thereby we describe a novel regime in the HSF-mediated protection against stress-induced protein damage.

cell biology↗

bioRxiv2018

CBP/EP300-dependent acetylation and stabilization of HSF2 are compromised in the rare disorder, Rubinstein-Taybi syndrome

Cells respond to protein-damaging insults by activating heat shock factors (HSFs), key transcription factors of proteostasis. Abnormal HSF protein levels occur in cancer and neurodegenerative disorders, highlighting the importance of the tight control of HSF expression. HSF2 is a short-lived protein, but it is abundant in the prenatal brain cortex and required for brain development. Here, we reveal that HSF2 is acetylated and co-localized with the lysine-acetyl transferases CBP and EP300 in human brain organoids. Using unbiased, biochemical, cell-imaging, and in silico approaches, we show that CBP/EP300 acetylates HSF2 at specific lysine residues, which promotes HSF2 stabilization, whereas the lysine deacetylase HDAC1 catalyzes its proteasomal degradation. The CBP KIX domain and KlX-recognizing motifs in HSF2 are critical for its interaction with acetylating enzymes. The functional importance of acetylated HSF2 is evidenced in Rubinstein-Taybi syndrome (RSTS), characterized by mutated CBP or EP300. We show that RSTS patient cells exhibit decreased HSF2 levels and impaired heat shock response. The dysregulated HSF pathway in RSTS opens new avenues for understanding the molecular basis of this multifaceted pathology.

developmental biology↗

bioRxiv2017

Integrated Computational Guide Design, Execution, And Analysis Of Arrayed And Pooled CRISPR Genome Editing Experiments

CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

molecular biology↗