The CRISPR Journal

The development of the central nervous system (CNS) depends on tightly regulated gene expression programs that guide neural progenitor differentiation and neuronal subtype specification. The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of nervous system development, with a larval CNS composed of just over 200 neurons and sensory cells. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona, validated single-guide RNAs (sgRNAs) have yet to be validated for key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected using CRISPOR and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates with predicted Doench 16 and Doench Ruleset 3 (RS3) scores, observing a modest but improved correlation with RS3 predictions. Based on these results, we recommend considering both scoring algorithms, with RS3 potentially offering improved predictive value for Ciona.

In gene editing, CRISPR/Cas approaches are often limited by off-target effects. In in vivo approaches involving multiple cell types, off-targets may result from unintended targeting of the wrong cells. In this work, we propose a solution to this limitation by using a transcribed intron of the target gene as an endogenous trigger (intron triggers) for a novel conditional guide RNA (intcgRNA). In vitro, intcgRNAs were responsive to the presence of the trigger. As a proof-of-concept, the human IL2 receptor subunit gamma gene (IL2RG) was then targeted using both the intcgRNA and the corresponding conventional crRNA in two cell lines: the lymphocytic HPB-ALL cell line, where IL2RG is highly expressed, and the epithelial HeLa cell line, where it is not. Sanger sequencing revealed that the crRNA and intcgRNA Cas9 complexes edited IL2RG with similar efficiency in HPB-ALL, whereas only the crRNA edited IL2RG in HeLa. This shows that intcgRNA avoids targeting unwanted cells that do not express the target gene, which is particularly relevant for in vivo targeting. The triggers of choice for conditional guides have been microRNAs, but as short intronic RNAs are far more diverse, trigger introns could become biomarkers of cell identity that improve the precision of CRISPR-based manipulations in vivo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/714022v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ae60cdorg.highwire.dtl.DTLVardef@1556c03org.highwire.dtl.DTLVardef@1264a0dorg.highwire.dtl.DTLVardef@c7d47d_HPS_FORMAT_FIGEXP M_FIG C_FIG

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

Accurate prediction of CRISPR-Cas9 guide RNA (gRNA) editing efficiency remains limited, particularly outside human systems, where models trained on exogenous human datasets show poor generalization. We analyzed Cas9 efficiency and repair outcomes using novel endogenous editing data from four human cell types, two tomato cell types, and cells from giant river prawn and black soldier fly. While integrating publicly available predictors via ensemble frameworks improved performance, our analysis revealed hundreds of novel features affecting activity. Crucially, dominant features related to sites competition for gRNA, and local geometric properties varied across systems, highlighting the strong context dependence of Cas9 efficiency and arguing against a universal model. Interestingly, codon usage bias-based features also emerged as informative predictors, as they are proxies for chromatin accessibility. In contrast, trends in repair outcomes remained conserved. This work provides essential resources for more generalizable CRISPR guide design.

The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive off-target assessment remains a bottleneck, particularly in large, complex genomes where existing tools often suffer from prohibitive computational costs, poor search-space convergence, and limited sensitivity toward non-canonical alignments involving insertions and deletions (indels). To address these limitations, we developed SpacerScope, an off-target analysis framework that enables unbiased, genome-wide discovery by leveraging binary vectorization and high-speed bitwise operations. Benchmarking against CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6,142/6,142) within our defined parameter space, achieving zero false negatives while identifying additional high-risk sites with complex edit distances. Unlike conventional tools such as Cas-OFFinder, CHOPCHOP, and CRISPOR, SpacerScope maintains high sensitivity for indel-inclusive off-targets that are otherwise overlooked. Our results establish SpacerScope as a high-speed, high-sensitivity solution for ensuring genome-editing specificity across diverse and complex genomic landscapes. The full source code, documentation, and multi-platform executables are available at https://github.com/charlesqu666/SpacerScope. Graphical TOCSpacerScope is a genome-wide off-target profiling tool for RNA-guided nucleases that combines binary vectorization, bitwise prefiltering, compact 2-bit substitution search, and right-end-anchored indel validation. In the evaluated datasets, it recovered all 6,142 parameter-defined true off-target sites from CIRCLE-seq and showed improved detection of indel-containing candidate sites relative to the previous tools tested here. SpacerScope provides interpretable event-level annotations and empirical risk scores for downstream guide assessment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/715005v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@bad2ddorg.highwire.dtl.DTLVardef@169b0aborg.highwire.dtl.DTLVardef@1cde3b2org.highwire.dtl.DTLVardef@1fbd11_HPS_FORMAT_FIGEXP M_FIG C_FIG Practitioner Points> SpacerScope enables genome-wide off-target profiling with explicit support for substitutions, insertions, and deletions in a single workflow. > The method combines binary-channel prefiltering with right-end-anchored validation to reduce the search space while preserving sensitivity within the evaluated parameter ranges. > SpacerScope returns event-level match annotations and empirical risk scores, supporting more interpretable downstream guide selection and benchmarking.

RNF43 is best known for removing the Wnt-receptor complex from the cell surface, thereby maintaining Wnt-signaling at minimal essential levels. Recent studies reported that RNF43-mutant colorectal cancers carrying the common BRAFV600E mutation, respond more effectively to combined BRAF/EGFR inhibition. To determine whether RNF43 directly regulates EGFR or BRAF protein abundance, multiple pancreatic and colorectal cancer cell line models were generated in which RNF43 was knocked out, repaired, or stably overexpressed. Total and cell surface EGFR levels, as well as endogenous BRAF expression, were quantified. Across all models, no consistent evidence emerges that RNF43 modulates endogenous EGFR or BRAF levels. R-spondins likewise fail to alter EGFR levels or internalization. Notably, elevated EGFR expression observed in a subset of RNF43 knockout clones is induced by unintended CRISPR/Cas9 vector integration rather than the absence of RNF43 itself, highlighting a previously underappreciated artefact that can confound interpretations of EGFR regulation in genome edited lines. Overall, the data argue against a direct and general role for RNF43 in controlling EGFR or BRAF protein abundance, contradicting recent reports that propose degradation of these targets. Further studies are required to resolve these discrepancies and clarify the mechanistic basis underlying these conflicting observations.

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

Pigs and chickens are not only the most important livestock species for global food production but also serve as key model organisms in various research disciplines. The pig is widely used in translational research due to its anatomical and physiological similarity to humans, providing valuable insights into immunology, metabolism, and disease mechanisms. In contrast, the chicken has become an essential model for studies related to poultry health, animal welfare, and developmental biology. Its externally developing embryo offers exceptional accessibility for experimental manipulation. Recent advances in genome editing technologies, particularly CRISPR/Cas9, have further expanded the potential of these species for functional genomic studies, although the efficient delivery of such tools remains a major challenge. By using virus-like particles (VLPs), we have been able to overcome this limitation. Here, we evaluated VLPs as delivery vehicles for genome engineering tools in pigs and chickens, two key livestock species at the human-animal interface. VLP-mediated delivery enabled efficient Cre recombination and high CRISPR/Cas9 editing rates in porcine cells, organoids, and oocytes, particularly when multiplexed. In chickens, VLPs supported robust Cre recombination and Cas9-mediated editing in cell culture, tracheal organ cultures, and in ovo. Reporter VLPs and dCas9 VLPs further demonstrated the versatility of this platform across porcine and avian systems. Together, these findings establish VLPs as an efficient and time-saving strategy for gene editing in livestock, with relevance for animal health, agricultural productivity, and translational One Health research.

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

The CRISPR-Cas9 system has revolutionized genome engineering, yet its full therapeutic potential remains constrained by challenges in precisely modulating its activity and specificity. Here we report a fully computational end-to-end pipeline for the de novo design of a single-domain VHH nanobody (NbSpCas9-v1) targeting a structurally conserved, non-catalytic epitope at the PAM-interacting (PI) and RuvC-III interface of Streptococcus pyogenes Cas9 (SpCas9; PDB: 4UN3). Nanobody sequences were generated using BoltzGen, a generative diffusion binder design framework, and co-folded with SpCas9 using Boltz-2 to evaluate structural confidence and binding affinity. The top-ranked model (SpCas9_4UN3_Bivalent_Hub_v1) achieved a complex pLDDT of 0.8406, an aggregate score of 0.8016, and an ipTM of >0.8, indicating high confidence in the nanobody-antigen interface. The designed 1,616-residue quaternary complex (SpCas9 + sgRNA + DNA + nanobody) was subjected to 10 ns of all-atom molecular dynamics (MD) simulation using the AMBER14SB force field within the GROMACS/OpenMM framework. The complex stabilized at RMSD [~]6 [A] with a radius of gyration of 39-44 [A], confirming thermodynamic stability under physiological conditions (310 K, 0.15 M NaCl). A conserved 96.3 [A] inter-molecular distance between the nanobody centroid and the HNH catalytic residue H840 establishes NbSpCas9-v1 as a distal, non-inhibitory binder -- ideally suited for a Bivalent Hub architecture recruiting secondary effectors to the Cas9 ribonucleoprotein (RNP). The nanobody-Cas9 interface is stabilized by 8 hydrogen bonds, 4 salt bridges, and [~]1,850 [A]2 of buried solvent-accessible surface area. These results provide a rigorous structural and dynamic foundation for experimental validation of VHH-based CRISPR-Cas9 enhancers and modulators. GRAPHICAL ABSTRACTThe computational workflow proceeds from SpCas9 crystal structure acquisition (PDB: 4UN3) through BoltzGen nanobody design, Boltz-2 structural co-folding, 10 ns explicit-solvent MD validation, and Bivalent Hub functional characterization. The PyMOL rendering below shows the full quaternary complex at atomistic resolution.

Long-read RNA sequencing (lrRNA-seq) provides advantages for transcript discovery and quantification through the sequencing of full-length transcripts. Although recent benchmarks have evaluated long-read technologies and quantification tools, to the best of our knowledge, no study to date has jointly compared sequencing technology, quantification choice, and depth across both bulk and single-cell platforms. Here, we generate a matched dataset using NGN2-induced neurons derived from Fragile X syndrome and isogenic rescue lines, profiled with bulk and single-cell Illumina, Oxford Nanopore Technologies (ONT), and Pacific Biosciences (PB) Kinnex technologies. All platforms and technologies capture the expected FMR1 reactivation signal. We find that PB bulk under-detects and under-quantifies short transcripts (less than 1.25 kb), ONT bulk under-detects and under-quantifies long transcripts (greater than 5 kb), and single-cell long-read technologies a large number of single-cell specific transcripts associated with truncations. Across six bulk and four single-cell long-read quantification tools, Isosceles, Miniquant, and Oarfish provide the best compromise between computational efficiency and performance in terms of quantification accuracy as measured by spike-ins, comparisons to Illumina, and on consensus based down-stream tasks such as differential transcript expression (DTE). Depth-equivalency analyses reveal that PB single-cell sequencing requires approximately three- to four-fold greater depth than bulk to reach comparable power for transcript discovery and differential transcript expression. Our work aims to offer practical guidance for study design, including the choice of technology, sequencing depth, and quantification method. In addition, we hope our data may serve a reference dataset to evaluate emerging long-read transcriptomic protocols and methods as well as more closely investigate FMR1 biology.

Cell and tissue-specific transcriptomic profiling of Caenorhabditis elegans is commonly achieved by fluorescence tagging or staining of targeted cell populations, often followed by fluorescence-activated cell sorting (FACS) and RNA sequencing. However, these approaches typically require separate strains for each labeled population, increasing labor and experimental variability while limiting direct comparison of multiple tissues within the same genetic background. To address this limitation and establish proof of concept, we engineered CELeidoscope, a multicolored C. elegans strain that enables spectral sorting of multiple major cell types within a single strain population. Strain construction was carried out using a high-throughput screening method that reduces the labor and plastic costs associated with transgene integration and outcrossing. Four primary tissues (body muscle, neurons, intestinal, and pharyngeal muscle cells) were tagged with spectrally distinct fluorescent proteins, allowing compatibility with viability and nucleic acid dyes. Using spectral flow cytometry, dissociated CELeidoscope cell suspensions could be sorted based on their spectral profiles, with cell recovery rates approximating the expected cell counts in whole organisms. Transcriptomic analysis of the sorted cell populations further validated the identity of the sorted populations, with recovered cells exhibiting gene expression signatures consistent with their intended cell and tissue identities. Together, these results establish CELeidoscope as a versatile tool for multiplexed cell-type isolation in C. elegans, providing a framework for tissue-specific analyses from a common strain background.

Gene drives are genetic elements that bias their own inheritance to spread desired traits in target populations, enabling population modification or suppression. Although homing-based drives can propagate efficiently, their potential for uncontrolled spread may present a challenge for field deployment. Thus, confined drive systems are needed. Here, we developed a confined modification drive, called Toxin-Antidote Recessive Embryo (TARE) drive, in the globally important malaria vector Anopheles stephensi. This drive works by cleaving and disrupting wild-type alleles in the germline or early embryo from maternally deposited Cas9. Disrupted alleles are recessive lethal, thus increasing the drive in a frequency-dependent manner. Inheritance bias was moderate in crosses between drive heterozygote mosquitoes, possibly due to low gRNA activity and thus moderate germline cleavage rates. Single-release cage trials confirmed the TARE drives ability to spread, although the drive ultimately declined due to fitness costs and resistance alleles associated with repetitive elements. Nonetheless our modelling analysis indicate that this TARE system could achieve population spread if the resistance issue is addressed. These findings demonstrate a functional prototype TARE drive in Anopheles stephensi and highlight key parameters governing its performance. Minor design optimizations could substantially improve efficiency and integrity, enabling rapid but confined population modification.

We present a novel integrative analysis of transposable elements (TEs) in 4 single cell RNA-seq (scRNA-seq) datasets of postmortem substantia nigra pars compacta samples of Parkinson Disease (PD) patients matched healthy controls, with the objective of building a cell-type specific trustworthy atlas of TEs that may clarify the role of TEs in sex differences in PD. We have used the soloTE tool to evaluate the TEs expression changes across all snRNA-seq studies identified in our previous systematic review, and then integrated the results using meta-analysis techniques. Finally, we evaluated the possible associations between TEs and protein coding genes by integrating our previous results in this matter with the information of TEs obtained, in order to propose the possible action mechanism by which some of the TEs contribute to PD.

Ehmt2 is a key H3K9 methyltransferase that regulates genome silencing and structural integrity during animal development. In addition to this canonical function, Ehmt2 has also been implicated in neural tissues mediating both direct and indirect transcriptional activation, and exon splicing, to facilitate proper neural cell differentiation and survival. Several germline loss-of-function animal models have been developed showing both conserved and divergent phenotypes that range from embryonic lethality to behavioural deficits in adult, fertile animals. Here, we generated the first maternal-zygotic ehmt2 loss of function mutant in zebrafish using CRISPR-Cas9 mutagenesis. An assessment of the pattern of H3K9 methylation in mutant embryos by ChIP-seq indicates that there are aberrant levels of this repressive mark, including reduction in discrete 5 non-coding regions of genes, but with no significant change in the overall pattern distribution of these marks across the genome. Global transcriptome and morphological analyses demonstrated that mutant embryos displayed greater variation in the timing of developmental progression that is, on average, slower compared to controls. Despite this, mutant embryos ultimately survive and are fertile. Through examination of progenitor cell dynamics and gene expression profiles, we found that the delay in embryonic development was associated with longer rates of S-M phases of the progenitor cell cycle in mutants leading to deficits in tissue growth. Finally, our data suggest a robust network of epigenetic regulators can potentially compensate for Ehmt2 loss of function and permit embryonic development and survival in ehmt2 mutant zebrafish. Our work establishes a zebrafish ehmt2 loss of function model that will facilitate examination of the complex and varied roles of Ehmt2 in vertebrate development.

MotivationThe ATHILA lineage of LTR retrotransposons has colonised all branches of the plant tree of life. In Arabidopsis thaliana and A. lyrata, ATHILA elements have invaded centromeres, influencing the genetic and epigenetic organisation, and driving satellite evolution. To assess the broader significance of ATHILA across plants, a computational pipeline is needed to identify ATHILA elements with high efficiency. Existing tools lack this ability because they are optimised for broad transposon classification at the expense of precise annotation of lower taxonomic levels. ResultsWe present ATHILAfinder, a pipeline for accurate and large-scale discovery of ATHILA elements. ATHILAfinder uses lineage-specific sequence motifs as seeds and additional filters to build de novo intact elements. Homology-based steps rescue intact ATHILA and identify soloLTRs. A detailed identity card includes coordinates, LTR identity, coding capacity, length and other sequence features for every ATHILA. We validate ATHILAfinder in the A. thaliana Col-CEN assembly and five additional Brassicaceae species, covering four supertribes and [~]30 million years of evolution. ATHILAfinder has very low false positive rates and outperforms widely-used tools like EDTA and the deep-learning-based Inpactor2 software for both recovery and precision of ATHILA. To demonstrate its usefulness, we generate insights into ATHILA dynamics across Brassicaceae. OutlookFew computational pipelines target specific transposon lineages, yet such tools can empower their identification and downstream analyses. Our tailored approach can be adapted to other LTR retrotransposon lineages, offering new ways for high-resolution analysis of transposons.

Bacteria can be engineered to express double-stranded RNA (dsRNA) that modulates eukaryotic host gene expression in a programmable manner via RNA interference (RNAi). This requires robust and systematic strategies for dsRNA circuit design and expression. Here, we developed modular genetic parts compatible with the CIDAR MoClo system for rapid assembly of dsRNA expression constructs in Escherichia coli HT115(DE3). We validated dsRNA production in vitro and assessed RNAi efficiency in Caenorhabditis elegans. A constitutive dsRNA circuit achieved rapid and near-complete gene knockdown, whereas a Ptac-driven circuit enabled tunable, partial silencing while minimizing the leakiness commonly observed in standard feeding RNAi systems. Together, this work expands the synthetic biology toolkit for dsRNA delivery, enabling precise control of RNAi outcomes from partial to complete gene silencing in nematodes.

CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Benchmarking on simulated datasets showed high accuracy, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling. Supplementary data are available online. AvailabilityALPINE is implemented in Python and distributed as Docker containers with Common Workflow Language (CWL) support for cloud deployment. The pipeline is available under MIT license at https://github.com/Maggi-Chen/ALPINE.

BackgroundPrecise control of DYRK1A dosage is essential for embryonic development, including craniofacial morphogenesis. While LZTS2 is among the most consistently identified DYRK1A-interacting proteins, its roles in embryonic development remain incompletely understood, and its potential contribution to craniofacial development has not been examined. Xenopus laevis was used to test the role of LZTS2 in craniofacial development and its functional relationship with DYRK1A. ResultsLzts2 and Dyrk1a showed overlapping expression during craniofacial development, with both proteins present in developing facial tissues. Knockdown of Lzts2 disrupted craniofacial morphogenesis and reduced expression of the neural crest-associated genes sox9 and pax3. These phenotypes closely resembled those caused by decreasing Dyrk1a function. Sub-phenotypic reductions of Lzts2 and Dyrk1a synergized to produce craniofacial defects, while partial reduction of Lzts2 attenuated aspects of the phenotype caused by Dyrk1a overexpression. Comparative analysis of human phenotypes associated with copy number gains of LZTS2 and DYRK1A revealed striking overlap, consistent with a potential functional interaction between these genes in humans. ConclusionsThese findings identify Lzts2 as a previously unrecognized regulator of craniofacial development and support a functional interaction with Dyrk1a during embryogenesis. Modulating LZTS2 or related regulatory partners may provide a strategy to selectively tune DYRK1A-dependent developmental pathways

Whole-exome sequencing (WES) enables the identification of rare germline variants contributing to pediatric diseases. Trio-based sequencing, comparing affected children with their parents, is particularly effective for rare disease genetics. However, WES data analysis requires bioinformatics expertise, varies across institutions, and is often incompatible with clinical workflows. We developed T-Rex (Trio Rare variant analysis of EXomes), a cross-platform desktop application that enables the standardized and local analysis of WES germline Trio data without the need for programming knowledge. T-Rex integrates state-of-the-art tools for alignment, dual-variant calling (GATK HaplotypeCaller + VarScan2), annotation (SNPEff/SNPSift), rare-variant filtering based on population frequencies (gnomAD), and family-based statistical testing, including the Transmission Disequilibrium Test with multiple-testing correction. Benchmarking of the dual-caller strategy on the Genome in a Bottle Ashkenazim Trio demonstrates high precision (99.2%) while maintaining robust sensitivity (91.1%). User testing (n=13) confirmed quick learning across clinicians and researchers. Application to a cohort of n=121 pediatric cancer Trio datasets, filtering for rare protein-coding variants (MAF[≤]0.1% in gnomAD v4.0), validated all assessable previously reported pathogenic variants. Overall, T-Rex enables clinicians to robustly analyze WES Trio data in compliance with data protection regulations without requiring additional software licenses. As one of the first platforms for comprehensive WES Trio analysis that requires no programming expertise while providing clinical-grade, end-to-end workflows, T-Rex facilitates collaborative research between clinics and reduces reliance on external providers. Implementation and AvailabilityThe source code is available on GitHub (https://github.com/SaraLuisaReh/trex). The fully precompiled app is available on Zenodo (https://zenodo.org/records/19135262).