Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enASCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A major contributor to this effect is gRNA representation bias within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also consider gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.

Background Genome editing in human skeletal muscle research requires protocols that maximize delivery while preserving viability and clonal outgrowth. We sought to develop a reagent-free workflow for CRISPR/Cas9 editing in human immortalized myoblasts and to demonstrate its performance in two use cases, an IARS1 knockout and an MLIP homozygous knock-in. Methods We optimized electroporation parameters using a green fluorescent protein reporter to compare three electrical settings for transfection and survival in E6/E7 myoblasts, then applied ribonucleoprotein delivery for editing. We evaluated the effect of confluency at electroporation, performed single-cell cloning without antibiotics or fluorescence-activated sorting, and validated edits by high-resolution melting pre-screen followed by Sanger sequencing. Results Electroporation optimization identified one parameter set that maximized delivery while preserving viability. Performing electroporation at low confluency increased clonal outgrowth and editing rates. The workflow yielded an 84% success rate for IARS1 knockout and a 3.3% success rate for MLIP homozygous knock-in. High-resolution melting provided a very sensitive pre-screen, detecting 96% to 100% of actual edits, reducing the number of Sanger sequencing needed. Performance was reproducible across runs and myoblast lines and increasing single-cell seeding scaled yields without compromising purity. Conclusions This work provides a practical and reproducible selection-free protocol that couples electroporation optimization, low confluency editing, single-cell cloning, and high-resolution melting sorting to generate pure edited myoblast lines. The approach is applicable to disease modeling in neuromuscular research and clarifies feasibility boundaries for essential genes and homology-directed repair in these cells.

Abstract The polycomb repressive complex 2 (PRC2) catalyses the addition of H3K27me3 marks to chromatin and thus modifies gene repression controlling the differentiation and function of medullary TECs (mTECs). The zinc finger protein AEBP2 physically interacts with PRC2 as a non-essential core component of the complex, yet its precise role in TEC biology remains untested. Here, we demonstrate that a TEC-targeted loss of AEBP2 expression in mice increases TEC and total thymic cellularity yet unexpectedly impairs the differentiation and maintenance of mimetic TEC subtypes. AEBP2-deficient mTECs display H3K27me3-dependent modifications in chromatin accessibility which compromises the capacity to promiscuously express tissue-restricted genes (TRGs). Consequently, severe lymphocytic infiltrates are observed in peripheral tissues as a result of flawed central T cell tolerance induction. Taken together, these findings highlight the essential, context-dependent role of AEBP2 in TEC differentiation and function via its impact on chromatin structure, transcriptional regulation, and developmental programming.

Abstract Non-coding RNAs represent a widespread and diverse layer of post-transcriptional regulation across cell types and states, yet much of their diversity remains uncharted at single-cell resolution. This gap stems from the limitations of widely used single-cell RNA-sequencing protocols, which focus on polyadenylated transcripts and miss many short or non-polyadenylated RNAs. Here, we adapted single-cell RNA-sequencing on the 10x Genomics platform to capture a broad complement of coding and non-coding RNAs—including miRNAs, tRNAs, lncRNAs, histone RNAs, and non-adenylated viral transcripts. This approach enabled the discovery of rich, dynamic non-coding RNA programs across immune cells, virally infected hepatocytes, and the developing human brain. In dengue virus-infected hepatocytes, we detect non-adenylated viral transcripts and distinguish active from transcriptionally quiescent infected states, each with distinct host regulatory signatures. In brain tissue, we identify biotype-specific, cell-type–restricted non-coding RNAs, including miRNAs whose expression anticorrelates with predicted targets, consistent with post-transcriptional regulatory relationships. We show that MIR137, one of the strongest GWAS loci associated with schizophrenia and intellectual disability, is expressed specifically in Cajal-Retzius cells, an early-born but transient population that guides subsequent cortical neuron migration. These findings demonstrate the importance of non-coding RNAs in defining cell identity and state, and show how expanded transcriptome coverage can reveal additional layers of gene control—now accessible through practical and scalable single-cell profiling.

Genes act within complex regulatory networks, and genetic variants can perturb these networks by altering gene co-expression. Here, we performed co-expression quantitative trait locus (co-eQTL) mapping using single-cell RNA-seq from the sc-eQTLGen consortium (1,330 donors, >2 million cells), enabling sensitive detection and prioritization of informative variant–gene–gene triplets. We identified co-eQTLs for 398 eGenes where a nearby genetic variant affected both the gene’s expression ( cis -gene) and its co-expression with other genes, often implicating upstream regulators. For 181 genes, we inferred a likely upstream transcription factor, with motif disruption predicted for 41 genes. These upstream genes are more often loss-of-function intolerant and show more network connections, providing an explanation for why co-eQTL variants are 2.8x more strongly associated with immune diseases than classical eQTLs. These findings position co-eQTLs as mechanistic links between genetic variation and disease, revealing how variants can rewire cell-type-specific gene networks.

ABSTRACT Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.

Ex vivo cell cultures are reductionist models that enable cost effective, precisely controlled experiments with fewer ethical concerns than in vivo conditions. This results in their extensive use in diverse studies, especially large-scale genetic and drug perturbation screens. Although it is commonly accepted that ex vivo models do not fully recapitulate in vivo conditions, the molecular effects of model systems on cells in homeostasis and on cells undergoing drug or genetic perturbations are poorly understood. Using a CRISPR knockout (KO) screen with transcriptome read-out (Perturb-seq) of hematopoietic progenitor cells cultured ex vivo and grown in vivo , we analyzed the effects of ex vivo culture on unperturbed and perturbed cells. Unperturbed cells cultured ex vivo generally showed reduced basal interferon signatures and increased growth and metabolism signatures. These differences in unperturbed cells translated to differences between KO effects observed in vivo and ex vivo . We validated this impact of the model system on KO effects in an additional dataset of genetic KOs in bulk-sorted splenic immune cells, which confirmed our results. Interestingly, genes and molecular pathways with different KO effects were partly predicted by differences between unperturbed cells cultured in vivo and ex vivo . We therefore evaluated the performance of state-of-the-art models in predicting in vivo KO effects from ex vivo KO effects. This proved challenging, demonstrating the need for further developments. In summary, our study reveals differences in culture models, suggests approaches to improve culture models, and provides a test case for computational predictions of perturbation effects.

Gene overexpression can be used to study gene function and is more suitable to characterize essential and redundant genes than gene knockout. A forward genetic approach based on random gene overexpression, also known as activation tagging , was previously used to study gene function in angiosperms. However, such an approach has never been applied to algae. Here, we present enhancer-driven random gene overexpression (ERGO), a forward genetic screen that we utilized to study genes involved in carotenoid metabolism in the green alga Chlamydomonas reinhardtii . We generated a library of over 33,000 insertional mutants in a yellow-in-the-dark background strain, which is incapable of producing chlorophyll in the dark. Each mutant contained a randomly inserted enhancer, E hist cons , capable of activating gene expression in the C. reinhardtii nuclear genome. After visually screening the mutant colonies for a color change from yellow to orange, we isolated a mutant with increased carotenoid content and remarkable resistance to high-light stress. RNA-seq data analysis revealed substantial upregulation of a gene, that we name CMRP1 , encoding a putative F-box protein. CRISPR-mediated knockout of this gene resulted in decreased carotenoid concentrations, confirming that CMRP1 is involved in the regulation of carotenoid metabolism. Our study shows that a gene overexpression screen can be successfully adapted to C. reinhardtii and potentially other plants and algae, thereby expanding the palette of genetic tools to study gene function.

In this study we demonstrate a previously uncharacterised post-translational regulatory mechanism governing flavivirus replication through the deubiquitylating enzyme ubiquitin C-terminal hydrolase L3 (UCHL3). Using activity-based protein profiling, we identified UCHL3 as a key cellular factor activated during Zika virus (ZIKV) and dengue virus (DENV) infections. CRISPR-Cas9 knockout experiments demonstrated that UCHL3 deficiency impairs flavivirus replication and viral protein expression across multiple cellular models. The underlying molecular mechanism involves UCHL3-mediated stabilisation of subgenomic flaviviral RNA (sfRNA)-containing biomolecular condensates. Through biotinylated sfRNA-interactome capture assays, we show that UCHL3 physically interacts with sfRNA-containing ribonucleoprotein complexes alongside G3BP1. Importantly, UCHL3 depletion triggers inappropriate RNase L activation, leading to sfRNA relocalisation from protective P-bodies to degradative compartments, such as RNase L-induced bodies (RLBs) as reported previously, resulting in viral RNA decay. Our rescue experiments confirmed that RNase L knockdown restores viral replication in UCHL3-deficient cells. This pro-viral effect of UCHL3 operates through interferon-independent mechanisms, as demonstrated by persistent replication defects even upon exogenous interferon treatment. This work therefore identifies UCHL3 as a molecular switch controlling the balance between pro-viral and antiviral RNA condensates, representing a promising host dependency factor for broad-spectrum flavivirus intervention strategies.

ABSTRACT During organogenesis, precise pre-mRNA splicing is essential to assemble tissue architecture. Many developmentally essential exons bear weak 5′ splice sites (5′SS) yet are spliced with high precision, implying unknown yet active splicing fidelity mechanisms. By combining transcriptome and alternative splicing profiling with temporal eCLIP mapping of RNA interactions across development, we identify the RNA-binding protein QKI as an essential direct regulator of splicing fidelity in key cardiac transcripts. Although QKI is dispensable for cardiac specification, its loss disrupts sarcomere assembly despite intact expression of sarcomere mRNAs through exon skipping and nuclear retention of mis-spliced RNAs. QKI-dependent exons in essential cardiac genes have weak 5′SS and frequently show poor complementarity with U6 snRNA. We show that QKI directly interacts with U6 snRNA using an overlapping interface to its traditional intronic binding activity, securing U4/U6·U5 tri-snRNP to ensure splicing fidelity. Thus, QKI exemplifies how context-aware RBPs enforce splicing fidelity at structurally vulnerable splice sites during organogenesis.

ABSTRACT Lipid droplets (LDs) are emerging as critical regulators of cellular metabolism and inflammation, with their accumulation in microglia linked to aging and neurodegeneration. Perilipin 2 (Plin2) is a ubiquitously expressed LD-associated protein that stabilizes lipid stores, and in peripheral tissues its upregulation promotes lipid retention, inflammation, and metabolic dysfunction. However, the role of Plin2 in brain-resident microglia remains undefined. Here, we used CRISPR-engineered Plin2 knockout (KO) BV2 microglia to investigate the contribution of Plin2 to lipid accumulation, bioenergetics, and immune function. Compared to wild-type (WT) cells, Plin2 KO microglia exhibited markedly reduced LD burden under both basal and oleic acid–loaded conditions. Functionally, this was associated with enhanced phagocytosis of zymosan particles, even after lipid loading, indicating improved clearance capacity in the absence of Plin2. Transcriptomic analyses revealed genotype-specific responses to amyloid-β (Aβ), particularly in pathways related to mitochondrial metabolism. Seahorse assays confirmed that Plin2 KO cells adopt a distinct bioenergetic profile, with reduced basal respiration and glycolysis but preserved mitochondrial capacity, increased spare respiratory reserve, and a blunted glycolytic response to Aβ. Together, these findings identify Plin2 as a regulator of microglial lipid storage and metabolic state, with its loss alleviating lipid accumulation, improving phagocytic function, and altering Aβ-induced metabolic reprogramming. Targeting Plin2 may therefore represent a potential strategy to modulate microglial metabolism and function in aging and neurodegeneration.

Small interfering RNAs (siRNAs) produced through the processing of viral double-stranded RNAs mediate potent antiviral RNA interference (RNAi) in eukaryotes. In Caenorhabditis elegans , such an antiviral defense is further amplified through the production of secondary siRNAs, yet the mechanisms by which secondary virus-derived siRNAs (vsiRNAs) confer protection remain poorly understood. Here, we characterize the role of rsd-6 , which encodes a Tudor domain protein and plays important role in antiviral RNAi, in vsiRNA biogenesis and modulation of viral pathogenesis. Using CRISPR Cas9-generated rsd-6 null mutants, we show that both primary and secondary vsiRNAs accumulate normally in the absence of RSD-6, indicating that it functions downstream of secondary vsiRNA biogenesis. We further showed that secondary vsiRNAs generated in rrf-1 -independent manner remained detected in the absence of RSD-6 and viral replication is further enhanced in rrf-1;rsd-6 double mutants compared to rrf-1 single mutants, suggesting a role of rsd-6 in mediating antiviral guided by all secondary vsiRNAs. Consistently, rsd-6 mutants exhibited more severe pathogenesis upon Orsay virus infection compared to rrf-1 mutants, underscoring its role as a major determinant of viral disease outcome. Domain characterization established that the N-terminal tandem domains of RSD-6 are required for antiviral activity, while the C-terminal Tudor domains are dispensable. Functional conservation was confirmed in C. briggsae , where silencing of the rsd-6 homolog enhanced viral replication. Together, our findings identify RSD-6 as a key effector acting downstream of secondary vsiRNA production and highlight its conserved role in modulating viral replication and pathogenesis across Caenorhabditis species. Importance In C. elegans , the RNAi-mediated antiviral defense relies on the production of secondary virus-derived siRNAs (vsiRNAs) to achieve an amplified antiviral effect. However, the mechanism by which these secondary vsiRNAs confer protection remains poorly understood. This is primarily due to the limited number of identified key effector genes. To address this knowledge gap, we profiled vsiRNA biogenesis in loss-of-function mutants and discovered that rsd-6 is dispensable for the production of secondary vsiRNAs, suggesting a role of rsd-6 in mediating antiviral defense downstream of secondary vsiRNA biogenesis. Worm survival assay further confirmed that rsd-6 is a critical modulator of viral pathogenesis and its antiviral function is conserved across Caenorhabditis species. The RSD-6 protein features three N-terminal tandem domains of unknow function and two tandem Tudor domains at its C-terminus. Our domain analyses demonstrated that the N-terminal tandem domains, but not the C-terminal Tudor domains, are essential for antiviral function. The identification of rsd-6 as a key effector gene acting downstream of vsiRNA biogenesis provides a solid foundation for elucidating the mechanism of antiviral RNAi amplification.

Iron-sulfur (Fe-S) clusters are essential cofactors required for the activity of numerous proteins involved in fundamental cellular processes, including DNA replication, metabolism and mitochondrial respiration. In eukaryotes, Fe-S cluster biogenesis is initiated in mitochondria by the ISC machinery, which assembles iron and sulfur, delivered by a cysteine desulfurase, onto the scaffold protein ISCU. Frataxin (FXN), a key regulator of this pathway, enhances Fe-S production by accelerating persulfide transfer to ISCU. FXN is essential in eukaryotes, and its loss results in “petite” phenotype in yeast, senescence in dividing mammalian cells and embryonic lethality in mice. Interestingly, in yeast, a methionine to isoleucine substitution at position 141 of the scaffold protein Isu1 can bypass the requirement of FXN. To test whether this bypass mechanism is conserved in mammals, we introduced the equivalent M141I substitution into the endogenous Iscu gene in murine fibroblasts carrying a conditional Fxn allele using CRISPR-Cas9. We show that the ISCU M141I variant enables cell survival in the absence of FXN, preventing cell cycle arrest and decreasing baseline DNA damage. However, these FXN-null survivor clones exhibit slower proliferation, persistent mitochondrial dysfunction and defective mitochondrial Fe-S cluster proteins. In contrast, nuclear and cytosolic Fe-S proteins are preserved, as is cellular iron homeostasis. Importantly, the ISCU M141I variant delays, but does not fully rescue, embryonic lethality in Fxn-deficient mice. Altogether, our results reveal a previously unrecognized compartment-specific rescue of Fe-S cluster dependent processes by the ISCU M141I variant in mammalian cells, raising for the first time the possibility of compartmental regulation of Fe-S cluster biogenesis.

ABSTRACT Transcription involves a cycle of initiation, pausing, elongation, and termination. SPT5 regulates both promoter proximal pausing and elongation, but how it orchestrates both steps during dynamic changes in gene expression remains unclear. Here, using Drosophila embryogenesis, we show that pausing both precedes and follows gene expression, while active transcription is accompanied by pause release. Optogenetic rapid depletion of SPT5 from the nucleus uncovered different sensitivities at different developmental stages. In early embryogenesis, SPT5 depletion caused a downstream shift in Pol II pausing to the +1 nucleosome, resulting in defective elongation and early termination. In late embryogenesis, it led to both up- and downregulation of expression, depending on the genes’ transcriptional and pausing state – upregulation is caused by pause release while downregulation is due to defective elongation. These changes are intensified when genes are increasing or decreasing their transcriptional state, indicating that SPT5 contributes to fine-tuning dynamic changes in gene expression.

This study presents the complete genome characterization of Lactiplantibacillus plantarum C6 a strain isolated from Indian dairy cheese using Illumina NovaSeq sequencing. The assembled genome (3.22 Mb, 44.5% GC) comprised 3076 coding sequences 59 tRNAs 10 rRNAs and 2 CRISPR arrays. Phylogenomic and ANI analyses confirmed its identity within the L. plantarum clade (>99% similarity with NMGL2 and DMDL 9010). Functional annotation revealed genes enriched in carbohydrate metabolism (10.7%) stress response and host-adaptation pathways supporting its probiotic potential. Bacteriocin biosynthetic gene clusters were identified, including those encoding PlnE PlnF PlnJ PlnK and PlnN indicating the strains ability to produce class II plantaricins. A RiPP cluster encoding a cyclic uberolysin-like peptide was also detected with structural similarity to known lanthipeptides such as Streptococcin A Nisin Q and Lacticin 3147 (Tanimoto scores 0.93 to 1.0) suggesting antimicrobial relevance. CAZy analysis revealed 102 carbohydrate-active enzymes (GHs, GTs) highlighting metabolic flexibility. To evaluate the antibiofilm potential of L. plantarum-derived metabolites 15 small molecules from cell-free supernatants (CFS) were selected through literature mining and subjected to molecular docking against the MRSA biofilm-associated enzyme poly-& 946;-1,6-N-acetyl-D-glucosamine synthase (encoded by icaA). 2,4 Ditert-butylphenol (-7.2 kcal/mol) and Indole-3-lactic acid (-7.1 kcal/mol) showed the strongest binding followed by Cyclo (L-propyl-L-valine) (-6.8 kcal/mol) and DL 4 Hydroxyphenyllactic acid (-6.4 kcal/mol) indicating promising inhibition of MRSA biofilm synthesis. Organic acids like acetic and lactic acid showed weaker interactions but may contribute synergistically through acidification. Overall L. plantarum C6 combines robust probiotic features genomic safety, and antimicrobial potential supported by bacteriocin gene clusters and effective antibiofilm metabolites highlighting its application in functional foods and novel antimicrobial development.

Summary Embryonic development is driven by dynamic protein networks, yet how these dynamics shape morphogenesis remains incompletely understood. Somitogenesis, the rhythmic segmentation of vertebrate embryos, is governed by signalling gradients and oscillations in the presomitic mesoderm (PSM) 1,2 , but the corresponding protein dynamics are largely unknown. Perturbations in this process cause congenital spine disorders and can result in embryonic lethality 3 . Here, we introduce an integrated proteomics and microfluidics approach to resolve spatiotemporal protein expression in the developing mouse tail. To this end, we established a microfluidic system to synchronize oscillations in embryo tails grown in 3D, which we combined with mass-spectrometry and RNA sequencing. We uncover novel oscillatory proteins and differentially expressed genes along the anteroposterior axis. Building on this dataset, we identify a previously unrecognized antagonistic, dynamic ligand–receptor expression pattern in R-Spondin/LGR signalling explaining how Wnt-oscillation amplitude increases despite decreasing ligand levels in anterior PSM. Dynamic ligand expression was validated in mouse gastruloids. Perturbation of ligand dynamics reduced oscillation amplitude and impaired somite formation. Our study reveals a novel regulatory strategy in which dynamic antagonistic gradients fine-tune signalling strength, providing new mechanistic insight into how protein dynamics control tissue patterning. We anticipate our dataset to serve as foundation for mechanistic investigations of mammalian somitogenesis including the role of mechanics and metabolism. More broadly, our approach combining microfluidics-based synchronization of signalling in multicellular systems with omics analyses can be applied to study dynamics in other contexts such as in tissue homeostasis 4 and regeneration 5 .

Abstract SUPT5H/SPT5, a universally conserved transcription factor across three domains of life, has been linked to promoter-proximal pausing, and its oncogenic role in cancer progression has been well-documented in several cancer types. In this study, we report CRISPR-Cas9-based SUPT5H knockdown-induced senescence as a potential cancer therapy for the first time. The knockdown of SUPT5H triggers the DNA damage-inducing p53 and Rb axes of senescence, leading to the upregulation of cell cycle inhibitors such as p21 and p27 resulting in inhibition of cell cycle progression proteins CDK4/6, as well as Cyclin D, E and A, thus resulting in senescence. Senescence induction results in the suppression of epithelial to mesenchymal transition via the upregulation of E-Cadherin and the downregulation of vimentin. Furthermore, the induction of senescence also leads to the suppression of immune evasion, offering a ray of hope in the fight against cancer.

Abstract Electronic biosensors offer a compact, integrable platform for real-time biomolecular detection, yet their performance is often limited by charge screening in physiological environments. Here, we report a porous ZnO/Al2O3 biosensor with grain boundary-engineered nanostructures that enables attomolar-level (0.5 aM) detection of microRNAs (miRNAs). The porous architecture forms spontaneously during thermal annealing through Zn2+ diffusion toward the Al2O3 layer along the ZnO grain boundaries and through lateral surface diffusion, generating localized porosity at structurally defined sites. These regions disrupt charge neutrality and introduce energy barriers, thereby enhancing surface potential shifts upon miRNA hybridization. The biosensor maintains high sensitivity in high-ionic-strength buffers, indicating effective suppression of Debye screening. Using the CRISPR-Cas13a system, we functionalized the surface with a crRNA targeting miR-17, thereby achieving sequence-specific detection and demonstrating compatibility with programmable molecular recognition. This grain boundary-guided nanostructuring strategy establishes a scalable route to engineering biosensors with localized sensitivity and target selectivity. Our approach provides a platform for ultrasensitive nucleic acid detection and offers strong potential for integration into miniaturized diagnostic systems.

We determined the potential of CRISPR/Cas13 technology as a therapeutic approach for centronuclear myopathies (CNMs) by reducing the expression of a single protein, DNM2. CNMs are severe congenital rare muscle disorders that result in muscle hypotrophy and weakness, with no cure. CNMs frequently result from mutations in either BIN1 , MTM1 , or DNM2 genes, with DNM2 being a key GTPase that plays a pivotal role in muscle membrane interactions with MTM1 and BIN1. Previous studies indicate that reducing DNM2 transcript expression by half could correct CNM phenotypes regardless the genetic forms, paving the way for a broad-spectrum CNM-therapy. We evaluated CRISPR/Cas13X.1-mediated DNM2 transcript knockdown, as a therapeutic application in a unique naturally-occurring canine CNM model harboring the DNM2 R465W /+ mutation, the most frequent pathogenic variant in patients. We show that in vivo intramuscular AAV-mediated CRISPR/Cas13X.1 injections, led to a reduction in DNM2 transcript and protein levels at one and two months post-treatment. Our results demonstrate the feasibility of CRISPR/Cas13-based therapy for CNM in a large animal model, paving the way for advancing this approach towards clinical trials.

Achieving scalable and sustainable production of cultivated meat hinges on developing robust livestock muscle and fat cell lines that can proliferate and differentiate effectively, while meeting regulatory standards and consumer expectations. In this study, bovine satellite cells (BSCs) were immortalized using CRISPR/Cas9 to knockdown PTEN (phosphatase and tensin homolog), TP53 (cellular tumor antigen) and SMAD4 (SMAD [Sma and Mad proteins] family member 4). The resulting cell line, named “CriBSC2,” exhibited consistent growth, maintained muscle cell characteristics, and successfully differentiated into multinucleated myotubes after more than 150 cell divisions. In contrast, another cell line, “CriBSC1,” achieved immortalization with TP53 knockout alone but lacked differentiation capacity. CriBSC2 were further cultured on gelatin scaffolds to evaluate their anchorage-dependent responses, laying the groundwork for their potential application in tissue engineering for cellular agriculture. Ultimately, CriBSC2 cells demonstrate suitable proliferation and differentiation capabilities crucial for advancing cellular agriculture and future food technologies.

Relapsed and refractory T cell malignancies are associated with poor clinical outcomes. Although autologous CAR-αβT cells have been employed in the treatment of several cancers, generating CAR-T cells for T cell malignancies remains challenging. The need to obtain enough healthy αβT cells from patients to generate CAR-αβT cells, combined with the problem of shared antigens such as the pan-cancer target CD38 expressed on both malignant and normal T cells, which can lead to fratricide of healthy CAR-αβT has hindered the application of CAR-T therapy for T-ALL. The use of allogeneic αβT and NK cells has also been explored for treating T-ALL. However deletion of the TCRαβ/CD3 complex to avoid graft-versus-host disease (GvHD) for αβT and multiple infusions of NK cells, can increase the cost of manufacturing and logistics. In this study, we genetically engineered polyclonal gamma delta T (γδT) cells as an alternative allogeneic cell source for cancer immunotherapy. γδT share innate immune properties of NK cells and adaptive immunity of αβT cells. Generating CAR γδT would allow us to minimize the cost of generating allogenic TRAC- KO CAR αβT cells and lower the number of infusions needed when NK cells are used. Utilizing a novel expansion protocol in combination with CRISPR/AAV gene editing, we developed CD38 knockout (CD38KO)/CD38-CAR polyclonal γδT cells that target T-ALL. Our editing strategy enabled site-directed, on-target insertion of the CD38-CAR transgene into the CD38 locus, with no evidence of significant random CAR DNA integration (as commonly seen with lentiviral CAR transduction) or chromatin abnormalities resulting from CRISPR editing. This approach effectively mitigated fratricide through simultaneous CD38 disruption and CAR expression. We evaluated the edited γδT cells in vitro across multiple patient-derived T-ALL samples and demonstrated the efficacy of the CD38KO/CD38-CAR γδT cells against both baseline and relapsed samples. In vivo, a single injection of CD38KO/CD38-CAR γδT cells without cytokine support resulted in potent anti-leukemic efficacy Fratricide-resistant CD38KO/CD38-CAR polyclonal γδT cells thus represent a promising off-the-shelf therapeutic platform. They may serve as a bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT), potentially enabling molecular remission in T cell malignancies and other CD38-expressing cancers such as AML.

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.

ABSTRACT Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7 , encoding a subunit of the mitochondrial F 1 F 0 ATP synthase, and overexpression of QDR2 and QDR3 , two previously uncharacterized members of the 12-spanner H⁺ antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a Δ FUM1 background strain improved titers of fumaric acid production from 0.26 g L −1 to 2.16 g L −1 . Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus . HIGHLIGHTS - Trifunctional CRISPR-AID enables gene activation, interference & deletion in K. marxianus . - Genome-wide CRISPR-AID screen identifies guides conferring fumaric acid tolerance. - verexpressing of QDR2 and QDR3 increases fumaric acid tolerance and production. - Deletion of mitochondrial gene ATP7 significantly improves fumaric acid tolerance. - Fermentation of QDR2 and QDR3 overexpression strain yields 2.16 g L⁻¹ fumaric acid.

Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.

CRISPR-Cas9 systems, adaptive defense mechanisms in bacteria and archaea, have been widely adopted as powerful gene editing tools, revolutionizing biological and medical research. In the first steps of CRISPR-Cas9 gene editing, the Cas9 protein, in complex with RNA, facilitates DNA melting and subsequent RNA-DNA hybrid formation, but the atomic-level mechanism of this fundamental process is not fully understood. Here, we present the results of long-timescale molecular dynamics simulations in which Cas9-RNA complexes bound to double-helical DNA and promoted the formation of RNA-DNA base pairs in a unidirectional, stepwise manner. Unexpectedly, we observed a direct role for the RNA in facilitating DNA melting events through a mechanism in which RNA bases intercalated within the DNA and promoted strand separation. In addition, breathing motions within the Cas9 DNA-binding cleft contributed to the sequential formation of RNA-DNA base pairs. These simulation results, obtained for two structurally distinct Cas9 proteins, together with supporting experimental work, suggest a novel RNA-dependent mechanism for DNA melting that may be conserved in other Cas proteins.