Polyglutamine (polyQ) diseases, including Huntington's disease and several spinocerebellar ataxias, are caused by abnormally expanded CAG nucleotide repeats, which encode aggregation-prone polyQ tracts. Substantial prior evidence supports a pathogenic role for polyQ protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. In this study, we developed a FRET-based reporter system that models polyQ aggregation in human cells and used it to perform a high-throughput CRISPR interference screen targeting all known molecular chaperones. This screen identified as a strong suppressor of polyQ aggregation the Hsp40 co-chaperone DNAJC7, which has previously been shown to modify aggregation of other disease proteins (tau and TDP-43) and has mutations causative for amyotrophic lateral sclerosis. We validated this phenotype and further established a physical interaction between DNAJC7 and polyQ-expanded protein. In contrast, DNAJC7 did not modify aggregation of polyglycine (polyG) in a FRET-based model of neuronal intranuclear inclusion disease. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in Mll2 -/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5’ untranslated regions (5’UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control.

Cardiac development is characterized by a complex series of molecular, cytoskeletal and electrophysiological changes that guarantee the proper functioning of adult cardiomyocytes (CMs). These changes are defined by cell-type-specific transcriptional rewiring of progenitor cells to form CMs, and are regulated by various epigenetic elements, such as long noncoding RNAs (lncRNAs). LncRNAs are versatile epigenetic regulators as they may act in cis or in trans to orchestrate important gene programs during cardiac development and may concurrently encode micropeptides. LIPTER is one such lncRNA, previously shown to regulate lipid droplet transport in cardiomyocytes and thus an important regulator of cardiomyocyte metabolism. Here we show that LIPTER also plays a role in the cytoskeletal maturation of CMs, as loss of LIPTER leads to persistent expression of fetal genes, changes in chromatin accessibility, disorganized sarcomeres and impaired calcium homeostasis in CMs. Furthermore, we have identified a cardiomyocyte-specific regulatory enhancer that regulates the expression of LIPTER in CMs. CRISPR-mediated inhibition of this enhancer led to reduced LIPTER expression in CMs and increased expression of fetal genes. This CM-specific enhancer could therefore be manipulated to control the expression of LIPTER for therapeutic benefit. In summary, we have unravelled a novel role of LIPTER in CMs cytoskeletal maturation and have identified a CM-specific enhancer for LIPTER.

Discovery of key growth drivers that can be targeted for therapy is a central goal in cancer research. While high-throughput CRISPR screens have revolutionized our ability to identify gene dependencies in cancer, most large-scale screens are conducted in two-dimensional (2D) culture systems that fail to recapitulate tumor organization and behavior. To uncover architecture-dependent vulnerabilities in breast cancer, we performed parallel CRISPR interference (CRISPRi) screens in 2D and three-dimensional (3D) cultures of MCF7 cells, an estrogen receptor-positive (ER+) breast cancer model representative of a high risk of relapse, luminal subtype. Knockdown of IFNAR2 and TYK2 conferred a growth advantage in 3D cultures, implicating type I interferon signaling as a tumor-intrinsic suppressor of proliferation in 3D spheroids. Transcriptomic and functional analyses demonstrated that type I IFN signaling is endogenously activated in 3D spheroids via RIG-I-mediated sensing of cytosolic double-stranded RNA, leading to TBK1 activation and induction of interferon-stimulated genes (ISGs). This tumor-intrinsic IFN response slowed proliferation in 3D culture, independent of exogenous stimuli or the presence of immune cells. Analysis of bulk, single-cell, and spatial transcriptomic datasets from breast cancer patients revealed that a subset of tumors exhibit elevated IFN signaling in cancer cells, including in immune-depleted tumor cores, consistent with a tumor-intrinsic IFN signature. Our findings uncover an IFN-mediated growth-suppressive program shaped by 3D tumor architecture, and contribute towards a better understanding of the role of tumor-intrinsic IFN activity.

Background Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP). Methodology/Principal Findings LAMP-CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the Leishmania ( Viannia ) subgenus, while the 18S assay showed pan- Leishmania detection capability. There was no cross-reactivity with other protozoan ( Trypanosoma cruzi and Plasmodium falciparum ) and bacterial ( Mycobacterium tuberculosis ) pathogen DNA, or with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% for kDNA and 72.7% for 18S rDNA, both with 100% specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load. Conclusions/Significance These new assays, being amenable to further simplification and optimization for their adoption in low-resource settings, hold promise as a new generation of accurate molecular tools for leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control. Author Summary Tegumentary leishmaniasis affects poverty-related populations in the Americas and encompasses skin and mucosal lesions that can cause disfigurement and social stigma. The disease is caused by several species of the protozoan parasite Leishmania. PCR-based molecular diagnostics are currently the most sensitive and specific diagnostic tools. Yet, these require specialized infrastructure and trained personnel that are not readily available in low-resource settings. New tools are required to meet the diagnostic needs in rural endemic areas. A promising tool leveraging CRISPR-Cas technology enables cost-effective, in vitro nucleic acid detection, paving the way for diagnostic solutions that could be made available to patients at, or near, the point of care. Here, we harnessed the CRISPR-Cas12a system combined with loop-mediated isothermal amplification (LAMP) to develop assays capable of detecting multiple Leishmania species of medical importance. Our assays employ multi-copy targets widely used in molecular diagnostics: the 18S rDNA for pan-Leishmania detection and a kDNA minicircle region conserved among L. (Viannia) species. Results can be read with either fluorescence detection or lateral flow strips. Both assays showed satisfying performance in both analytical validation and clinical sample testing under laboratory conditions. These new tools show promise to improve diagnosis and surveillance of leishmaniasis.

Multiplexed methods for nucleic acid detection are immensely challenging to deploy outside of laboratory settings. Conversely, field-deployable methods are limited to low levels of multiplexing. During the COVID-19 pandemic, we developed Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a sensitive and deployable CRISPR-based technology for nucleic acid detection. Here, we introduce microfluidic SHINE (mSHINE) which enables >100-plex nucleic acid detection using a highly portable microfluidic manifold. The manifold directs a diluted sample into individual reaction chambers, each of which contains lyophilized SHINE reagents and a microscopic stir bar or bead for mixing. Samples can be loaded using a syringe by hand, greatly simplifying the testing process. A subsequent sealing step allows for >100 SHINE reactions to proceed independently and in parallel. We demonstrate that mSHINE has equal sensitivity to SHINE, allowing for highly multiplexed pathogen detection in ≤ 1 hour. In addition, mSHINE can detect single-nucleotide variants, including mutations associated with drug susceptibility. mSHINE shifts the paradigm of laboratory-based multiplexed nucleic acid testing, greatly benefiting patients and public health.

ABSTRACT Pseudomonas aeruginosa is a metabolically versatile opportunistic human pathogen. It causes acute and chronic infections and is notorious for its multidrug resistance and tolerance. To systematically uncover genetic vulnerabilities that could be exploited as therapeutic targets, we present a portable high-density CRISPR interference (CRISPRi) library comprising >80’000 single-guide RNAs (sgRNAs) targeting virtually all annotated coding sequences and intergenic regions of P. aeruginosa PAO1. This library was used to assess the genome-wide fitness landscapes under different growth conditions, uncovering gain- and loss- of-function phenotypes for more than a thousand genes upon depletion. Many of the phenotypes are likely caused by hypomorphic (partial loss-of-function) alleles that would not be easily accessible by traditional transposon sequencing (Tn-Seq). Focusing on central carbon metabolism, we reveal two glyceraldehyde-3-phosphate dehydrogenases as central, non-redundant nodes in glycolytic and gluconeogenic growth conditions that might be promising targets to redirect carbon flux away from metabolically persistent states associated with chronic infections. More generally, our comprehensive sgRNA libraries are a valuable resource to access genome-wide quantitative phenotypes through CRISPRi beyond the binary phenotypes offered by Tn-Seq.

ABSTRACT Acral melanoma (AM) is an aggressive melanoma subtype with limited therapeutic options and poor outcomes. In non-European descent and admixed populations, like those residing in Latin America, AM accounts for a significant proportion of cutaneous melanoma cases. Here, we performed comprehensive genomic and functional profiling of AM from a uniquely diverse Brazilian cohort. Whole-exome and transcriptome sequencing revealed low mutation burden and predominance of copy number alterations, including high-amplitude focal amplifications termed hailstorms. These hailstorms frequently affected chromosomes 11, 5 and 22 and key oncogenes such as CCND1 , GAB2 , CDK4 , and TERT . The presence of hailstorms in the long arms of chromosomes 11 and 22 was associated with higher focal copy number burden and loss of DNA damage response genes ( ATM , CHEK1 ), suggesting a permissive genomic environment driving structural instability. To explore the unique genomic context of AM, we established a comprehensive collection of patient-derived xenograft (AM-PDX) models that faithfully retain the histopathological and genomic features of the original tumours. Functional exploration of AM-specific vulnerabilities through pharmacological and CRISPR/Cas9 knockout screenings identified strong sensitivity to targeting MAPK, CDK4/6, MDM2, and WEE1 pathways. Notably, the pan-RAS(ON) inhibitor RMC-7977 effectively reduced viability in NRAS -, KRAS -, and KIT -mutant AM cell lines. Finally, CRISPR screens revealed dependencies selectively essential in AM, including CRKL and SF3B4 , highlighting previously unrecognized vulnerabilities. Our findings emphasize the distinct biology of AM compared to other subtypes of melanoma, provide a valuable resource of models reflective of Latin American ancestry, and identify potential drivers and therapeutic targets.

Closely related species often exhibit distinct morphologies that can contribute to species-specific adaptations and reproductive isolation. One example are Lepidopteran caterpillar appendages, such as the “caudal horn” of Bombycoidea moths, which have evolved substantial morphological diversity among species in this group. Using interspecific crosses, we identify the genetic basis of the caudal horn size difference between Bombyx mori and its closest relative B. mandarina . The three largest of eight QTL account for one third the mean horn length difference between the species. The largest of these, on chromosome 4, encompasses a conserved Wnt family gene cluster, key upstream regulators that are well-known for their roles in morphological diversification in animals. Using allele-specific expression analysis and CRISPR/Cas9 knockouts, we show that tissue-specific cis -regulatory changes to Wnt1 and Wnt6 contribute to the species difference in caudal horn size. This kind of modularity enables highly pleiotropic genes, including key upstream growth regulators, to contribute to the evolution of morphological traits without causing widespread deleterious effects. Significance This study explores the genetic basis of a distinct morphological trait that varies between two closely related moth species, providing insights into the evolution of morphological diversity. By identifying cis -regulatory changes in two Wnt family genes as major contributors, this work underscores the importance of developmental gene regulatory networks in shaping species-specific traits. The findings illustrate how even small modifications in major upstream regulator genes can drive significant phenotypic variation, revealing how genetic changes in key growth regulators fuel the diversification of form and function. These results advance our understanding of the mechanisms behind the evolution of complex morphological traits.

Abstract Background The behaviour of complex biological systems emerges from the coordinated activity of networked molecular components. In this context, gene regulatory networks (aka gene coexpression networks) offer insights into the regulation of gene expression programs. In cancer, aberrant gene expression underlies molecular and clinical features, and identifying key networked transcriptional regulators may enable targeted therapeutic interventions. However, computationally inferred regulatory nodes have so far hardly been experimentally validated. Results Here we combined gene expression network analysis with gene perturbation experiments to test whether computationally identified hub genes act as upstream regulators of their coexpression modules in breast cancer. To better capture the context-dependent nature of gene regulation and minimize confounding effects due to heterogeneity, we also constructed subtype-specific networks. Using the METABRIC transcriptomic dataset of primary breast tumours, we identified clinically-informative gene modules in the highly aggressive basal-like subtype. Candidate regulatory hubs were prioritized based on network centrality, and their functional relevance was assessed both in silico and in vitro . CRISPR-mediated knockout of selected hub genes resulted in coordinated down-regulation of module genes and impaired cellular functions, demonstrating causal links between hub gene function, module expression and phenotypic outcome. Moreover, we observed a significant correlation between the transcriptional impact of each knockout and its functional effects—highlighting the biological relevance of coexpression modules and supporting the hypothesis that their structure reflects functional dependencies. Conclusions To our knowledge, this is the first study to functionally validate clinically relevant hub genes, providing direct support for the predictive power of coexpression-based network models.

Abstract Midbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond.

White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and water-borne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures, such as augmented biosecurity, selective breeding, and immunostimulants, have, despite extensive research, achieved only limited success. New biotechnological tools, such as RNA interference, CRISPR-Cas gene editing, and nanotechnology, offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.

CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.

ABSTRACT Chromatin is dynamic at all length scales, influencing chromatin-based processes, such as gene expression. Even large-scale reorganization of whole chromosome territories has been reported upon specific signals, but lack of suitable methods has prevented analysis of the underlying dynamic processes. Here we have used CRISPR-Sirius for time-lapse imaging of chromatin loci dynamics during serum starvation. We show that the chromosome 1 loci move towards the nuclear envelope during the first hour of serum starvation in a chromosome-specific manner. Machine learning-assisted exploration of acquired multiparametric data combined with the Shapley values-based explanation approach allowed us to uncover the critical features that characterize chromatin dynamics during serum starvation. This analysis reveals that although serum starvation affects overall nuclear morphology and chromatin dynamics, chromosome 1 loci display a specific response that is characterized by maintenance of dynamics in constrained environment, and long “jumps” at the nuclear periphery. Interestingly, the two homologous chromosomes display differential behaviors, with the more peripheral homolog being more responsive to the signal than the internal one. Overall, the presented machine learning-assisted dataset exploration helps us navigate the multidimensional data to understand the underlying dynamic processes and can be applied to a wide variety of research questions in imaging and cell biology in general.

CRISPR/Cas9 has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profiled CRISPR/Cas9 editing outcomes at 95 loci using our established CRISPR/Cas9/AAV9-sgRNA system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR/Cas9-mediated editing in vivo largely align with those observed in vitro but with reduced editing precision. Additional to the anticipated small editing indels such as MMEJ mediated deletions and NHEJ mediated templated insertions, we uncovered a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by three-dimensional chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo , we identified their distinct roles in regulating the large on-target modifications. Together, our work for the first time systematically profiles the CRISPR/Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR/Cas9-based gene therapies.

Deciphering spatiotemporal cell lineage dynamics remains a fundamental yet unresolved challenge. Here we introduce eTRACER, a novel CRISPR-Cas9 lineage tracer that targets neutral 3’UTR of high-expression endogenous genes, enabling efficient recovery of static and evolving barcodes from single-cell and spatial transcriptomics. By optimizing gradient editing efficacy and avoiding large disruptive deletions, eTRACER reconstructs high-fidelity and high-resolution single-cell phylogenies. Applied to EGFR-mutant lung adenocarcinoma (LUAD) under CD8 + T cell cytotoxicity, eTRACER reveals directional state transitions from Hypoxic and Proliferative states to Epithelial-Mesenchymal Transition state during immune evasion. Spatially-resolved lineage mapping unveils layered stratification of distinct tumor states and location-primed cell migration and state transitions. Lineage-coupled single-cell multiomic analysis uncovers cooperative mechanism between tumor cell-intrinsic AP-1 transcriptional program and spatially restricted macrophage-tumor cell interaction leading to immune evasion. Collectively, we develop a powerful spatiotemporal lineage tracer and uncover microenvironment-primed cellular evolution underlying immune evasion of EGFR-mutant LUAD, with important implication for efficient immunotherapy.

Reverse genetics, facilitated by CRISPR technologies and comprehensive sequence-indexed insertion mutant collections, has advanced the identification of plants genes essential for arbuscular mycorrhizal (AM) symbiosis. However, a mutant phenotype alone is generally insufficient to reveal the specific role of the protein in AM symbiosis and in many cases, identifying interacting partner proteins is useful. To enable identification of protein:protein interactions during AM symbiosis, we established a Medicago truncatula -Diversispora epigaea yeast-two-hybrid (Y2H) library which, through Y2H-seq screening, can provide a rank-ordered list of candidate interactors of a protein of interest. We also developed a vector system to facilitate bimolecular fluorescence complementation assays (BIFC) in mycorrhizal roots so that protein interactions can be assessed in their native cell types and sub-cellular locations. We demonstrate the utility of a Y2H-seq screen coupled with BIFC in mycorrhizal roots, with a search for proteins that interact with CYCLIN DEPENDENT LIKE KINASE 2 (CKL2), a kinase essential for AM symbiosis. The Y2H-seq screen identified three 14-3-3 proteins as the highest ranked CKL2 interacting proteins. BIFC assays in mycorrhizal roots provided evidence for a CKL2:14-3-3 interaction at the periarbuscular membrane (PAM) in colonized root cells. Down-regulation of 14-3-3 by RNA interference provides initial evidence for a function in AM symbiosis. Thus, CKL2 may utilize 14-3-3 proteins to direct signaling from the PAM. The Y2H and BIFC resources will accelerate understanding of protein functions during AM symbiosis.

Strigolactones are ecologically, developmentally, and physiologically important hormones, but much remains unknown about their evolution and role in non-model species. Sorghum is an important C 4 cereal for ∼1 billion people globally and exhibits natural variation in root-exuded strigolactones. Differences in sorghum strigolactone stereochemistry are associated with resistance to a parasitic plant, but with evidence for potential trade-offs. In the present study, we studied sorghum mutants of loci in the strigolactone biosynthetic pathway, C AROTENOID CLEAVAGE DIOXYGENASE 8 ( CCD8 ) and LOW GERMINATION STIMULANT 1 ( LGS1 ). We found that CCD8 CRISPR-Cas9 deletions changed the accumulation of low abundance metabolites, reduced net carbon assimilation rate, altered root architecture and anatomy, and reduced the establishment and benefit of mycorrhizal symbionts. For LGS1 CRISPR-Cas9 deletions, we found net carbon assimilation rate to be reduced, the colonization of mycorrhizal symbionts to be delayed, and evidence for regulatory pathways involved in stress response and growth to be impacted. We further tested the impacts of restoring functionality of LGS1 into a normally non-functional background (RTx430). Notably, we did not see consistent impacts of LGS1 loss-of-function across LGS1 deletion and insertion mutants, though root exudates from insertion mutants increased stimulation of Striga germination, suggesting that background specific modifiers may buffer the strigolactone impacts of loss-of-function at LGS1 . Our study begins to give context to the trade-offs associated with a host resistance strategy to a parasitic plant and more broadly contributes to understanding the role strigolactones play in sorghum physiological processes, growth, and development.

ABSTRACT Background The histone chaperone complex, consisting of the death domain–associated protein (DAXX) and the alpha-thalassemia/mental retardation X-linked protein (ATRX), plays a pivotal role in maintaining chromatin through the deposition of the histone variant H3.3. Mutations leading to loss of ATRX or DAXX function are linked to the non-telomerase, alternative lengthening of telomeres (ALT) phenotype in certain cancers. Engineered ATRX mutations have previously been found to induce features of ALT in prostate cancer cell lines, notably in LAPC-4, but not in CWR22Rv1. This study determined the impact of DAXX mutations on ALT-associated characteristics in CWR22Rv1 and LAPC-4. Methodology Mutations were induced in CWR22Rv1 and LAPC-4 cells by targeting exon 2 of DAXX using the CRISPR-Cas9 genome editing strategy. The resulting mutant clones were then evaluated for ALT-associated characteristics, including the presence of ALT-associated PML bodies (APBs), C-circles, telomere length heterogeneity, and a lack of telomerase activity. Results Four CWR22Rv1 DAXX mutant clones ( DAXX Mut 1 - 4 ) and five LAPC-4 clones ( DAXX Mut 1-5 ) were evaluated. In CWR22Rv1, DAXX Mut 1, DAXX Mut 2, and DAXX Mut 4 were true knockout clones with frameshift mutations in both copies, while CWR22Rv1 DAXX Mut 3 had a frameshift mutation in one copy and an in-frame mutation in the other. Protein expression was undetectable in all the CWR22Rv1 clones, including CWR22Rv1 DAXX Mut 3 . In LAPC-4, DAXX Mut 1 was a true knockout, while DAXX Mut 2, DAXX Mut 3, DAXX Mut 4 , and DAXX Mut 5 clones had at least one in-frame mutation. Among these LAPC-4 clones, only DAXX Mut 1 had undetectable protein by western blotting. ALT-associated characteristics such as APBs, C-circles, and telomere length heterogeneity were observed only in CWR22Rv1 DAXX Mut 4 . All the clones maintained telomerase activity, regardless of whether ALT-associated hallmarks were observed. Implications The CWR22Rv1 and LAPC-4 DAXX mutant clone models provide useful tools for future studies on telomere maintenance mechanisms and DAXX-related biology, particularly in prostate cancer.

Abstract Here we report unprecedented efficacy in the functional repair of an exemplar locus, ornithine transcarbamylase (OTC), in mutant primary mouse and human hepatocytes in vivo using a dual AAV vector system configured to deliver CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration by non-homologous end joining. The approach was mutation agnostic and targeted editing events to intronic sequences to prevent inadvertent inactivation of hypomorphic alleles. Notably, in a murine model, we corrected the metabolic defect and simultaneously achieved liver-wide restoration of physiological metabolic zonation of OTC expression by capture of the native promoter. The effectiveness of this approach was confirmed using a universally configured therapeutic cassette in patient-derived primary human hepatocytes in vivo. These data provide a powerful template to guide further optimization of this approach and, given the high editing efficacy required for phenotypic effect in OTC deficiency, have broader relevance to other liver disease phenotypes.

Abstract We have developed a CRISPR/Cas based assay able to distinguish between two ranges of closely related RNA targets using two detection channels. This required a pipeline to design RNA guide sets with the right degree of specificity. We tested our approach using SARS-CoV-2 and zoonotic near-neighbor sarbecoviruses. Using pre-existing guide design rules, we utilized a machine learning based model to design and optimize guide sets for specific detection of SARS-CoV-2 and separately to its nearest neighbors. The in vitro testing of the guide sequences has shown that Cas13 assays can tolerate more mismatches than assumed based on previous guide design rules. Mismatches located closer to the 3’ end of the guide and mismatches evenly distributed throughout the guide resulted in a smaller impact on the guide’s ability to activate the Cas enzyme. Modified SHERLOCK assay for detection and discrimination of SARS-CoV-2 and its zoonotic coronaviruses was developed using optimized sets of guides. The final assay was able to classify the targets into three classes 1) SARS-Co-V2, 2) closest known SARS-Co-V2 near-neighbor BANAL-236 and 3) the remaining zoonotic near-neighbors. This approach provides value through early detection of natural and engineered variants.

Despite the growing catalog of long noncoding RNAs (lncRNAs), the functional roles of their vast majority in cancer remain poorly defined. To systematically explore lncRNA dependencies in triple-negative breast cancer (TNBC), we compiled a comprehensive annotation by merging GENCODE, BIGTranscriptome, and MiTranscriptome databases and performed a CRISPR-Cas9 deletion screen targeting 1,029 TNBC-enriched lncRNAs. The screen revealed several essential lncRNAs and those modulating doxorubicin sensitivity, with TPL1 emerging among top hits. TPL1 silencing significantly impaired TNBC cell proliferation in both 2D and 3D cultures and reduced invasive capacity in an organ-on-chip model. Transcriptomic and proteomic profiling following TPL1 knockdown revealed downregulation of genes involved in ECM–receptor interaction, focal adhesion, cell migration, and PI3K-Akt signaling. Mechanistically, TPL1 directly interacted with key proteins including EIF4B, MDM2, TARBP2, TLE5, and GTPase RAN, suggesting TPL1 could regulate RNA processing, transcriptional repression, and translation, as well as modulate GTPase signaling pathways. Additionally, TPL1 functioned as a competing endogenous RNA (ceRNA), sequestering miR-10396b-5p, miR-486-3p, and miR-450a-2-3p, among others, thereby modulating expression of pro-tumorigenic targets. Clinically, TPL1 was significantly overexpressed in TNBC tissues, particularly in the BLIS subtype. Collectively, our findings highlight TPL1 as a key regulator of TNBC molecular networks and a promising therapeutic target.

Precise regulation of transcription factor (TF) expression is critical for maintaining cell identity, but studies on how graded expression levels affect cellular phenotypes are limited. To address this gap, we employed human embryonic stem cells (hESCs) as a dynamic model to study gene dosage effects and systematically titrated key TFs NANOG and OCT4 expression using CRISPR interference (CRISPRi). We then profiled transcriptomic changes in hESCs under self-renewal and differentiation conditions using single-cell RNA-seq (scRNA-seq). Quantitative modeling of these Perturb-seq datasets uncovers distinct response patterns for different types of genes, including a striking non-monotonic response of lineage-specific genes during differentiation, indicating that mild perturbations of hESC TFs promote differentiation while strong perturbations compromise it. These discoveries suggest that fine-tuning the dosage of stem cell TFs can enhance differentiation efficiency and underscore the importance of characterizing TF function across a gradient of expression levels.

Cyclin-Dependent Kinase 4 (CDK4) is a key regulator of cell cycle progression, driving the G0/G1-to-S phase transition through phosphorylation of Retinoblastoma 1 (RB1). Clinically, CDK4/6 inhibitors are under investigation in Triple Negative Breast Cancer (TNBC), a subtype characterized by invasiveness, aggressiveness and poor prognosis. While CDK4 is primarily targeted for its role in proliferation, emerging evidence suggests it may also regulate other cellular processes. In particular, the mechanisms by which CDK4 could influence cancer cell migration, remain largely unexplored, particularly in highly heterogenous cell line like MDA-MB-231. This study investigates whether CDK4 contributes to the regulation of TNBC cells migration and identifies the pathways involved in MDA-MB-231 cells, independently of its role in proliferation. We demonstrate that loss or inhibition of CDK4, using respectively CRISPR/Cas9 mediated CDK4 knockout and pharmacological CDK4/6 inhibitor, leads to enhanced migration capacities and reorganization of actin subcellular networks. Mechanistically, the absence of CDK4 results in decreased phosphorylation of Myo9b at serine 1935 (S1935), which enhances RhoA signaling, a key driver of cytoskeletal dynamics, leading to polarity defects and increased cell migration. These findings reveal a non-canonical function of CDK4 in limiting TNBC cell migration through the CDK4/CyclinD-Myo9b-RhoA signaling axis. This work highlights the broader cellular roles of CDK4 beyond its established function in proliferation and suggest that inhibition of Myo9b-RhoA pathway could reduce metastatic behaviour in TNBC treated with CDK4/6i, thereby informing future co-therapeutic strategies against aggressive cancer subtypes.

Summary The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.