Abstract Bialleleic pathogenic variants in LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report the use of gene editing to generate isogenic LCA5 knock-out (LCA5 KO) induced pluripotent stem cells (iPSC) and their differentiation to retinal organoids. The molecular and cellular phenotype of the LCA5 KO retinal organoids was studied in detail and compared to isogenic controls as well as patient-derived retinal organoids. The absence of LCA5 was confirmed in retinal organoids by immunohistochemistry and western blotting. There were no major changes in retinal organoid differentiation or ciliation, however, the localisation of CEP290 and IFT88 was significantly altered in LCA5 KO and patient photoreceptor cilia with extension along the axoneme. The LCA5-deficient organoids also had shorter outer segments and rhodopsin was mislocalised to the outer nuclear layer. We also identified transcriptomic and proteomic changes associated with the loss of LCA5. Importantly, treatment with the small molecules eupatilin, fasudil or a combination of both drugs reduced CEP290 and IFT88 accumulation along the cilia. The treatments also improved rhodopsin traffic to the outer segment and reduced mislocalisation of rhodopsin in the outer nuclear layer. The improvements in cilia-associated protein localisation and traffic were accompanied by significant changes in the transcriptome towards control gene expression levels in many of the differentially expressed genes. In summary, iPSC-derived retinal organoids are a powerful model for investigating the molecular and cellular changes associated with loss of LCA5 function and highlight the therapeutic potential of small molecules to treat retinal ciliopathies.

ABSTRACT A novel therapeutic strategy was recently proposed for high-risk neuroblastoma carrying copy number gain of the TRIM37 gene: centriole loss upon inhibition of polo-like kinase 4 (PLK4), while tolerated by normal cells, induces aberrant mitotic spindle formation and p53-dependent cell death in TRIM37 -overexpressing cells. Interestingly, while full PLK4 inhibition causes centriole loss, partial inhibition is known to elevate centriole numbers. Here we show using a novel selective PLK4 inhibitor RP-1664 that both centriole loss and amplification contribute to hypersensitivity of neuroblastoma cells. Whereas inactivation of TRIM37 and TP53 rescues neuroblastoma cell death at higher concentrations of RP-1664, at lower doses cell death is TRIM37/TP53 -independent. With CRISPR screens and live cell imaging we demonstrate that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to inability to cluster or inactivate supernumerary centrosomes. In vivo , RP-1664 shows robust efficacy in neuroblastoma xenografts at doses consistent with centriole amplification. STATEMENT OF SIGNIFICANCE High-risk neuroblastoma is associated with poor outcomes in pediatric patients and novel therapies need to be developed. We show that neuroblastoma cells are remarkably sensitive to PLK4 inhibitors due to a combination of two complementary mechanisms, supporting the evaluation of PLK4 inhibitors in clinical trials of high-risk neuroblastoma.

ABSTRACT In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single cell migration. Polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is well established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here we performed a screen for novel regulators of collective cell migration by identifying genes associated with the RAC1-WAVE-Arp2/3 pathway at cell junctions using dependency maps and by inactivating these candidates using CRISPR/Cas9. In wound healing, MCF10A epithelial cells collectively migrate towards the free space in a coordinated manner. PKN2 knockout (KO) cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a swirling behavior. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of cells migrating towards the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1 and their phenotype is rescued by constitutively active YAP1. Together, our data reveal two complementary activities required for coordinating cells in collective migration.

ABSTRACT The construction of genome-wide DNA libraries from publicly available resources is essential for leveraging functional genomics to investigate complex biological systems. However, all existing high-throughput cloning methods for transferring DNA fragments between vectors require PCR amplification of the DNA fragments, rendering the construction of genome-wide DNA libraries labor-intensive and time-consuming. By introducing a concept of CRISPRshuttle cassette, we herein present a method named CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method enables the high-throughput transfer of numerous DNA fragments from original plasmids with identical backbones to a different vector background without the need for PCR amplification of the DNA fragments. The procedure comprises two-step test tube reactions followed by bacterial transformation. Using CRISPRshuttle we successfully generated a library of GAL4/UAS-based UAS-ORF plasmids covering 1,397 human genes conserved in Drosophila . This library may serve as a valuable resource for gain-of-function screening in cultured cells and for the creation of a transgenic UAS-ORF library in Drosophila .

The complexity of molecular discovery requires autonomous systems that efficiently explore vast and uncharted chemical spaces. While integrating artificial intelligence (AI) with robotic automation has accelerated discovery, its application remains constrained in fields with scarce historical data. One such challenge is the design of lipid nanoparticles (LNPs) for mRNA delivery, which has relied on expert-driven design and is hindered by limited datasets. Here, we introduce LUMI-lab, a self-driving lab (SDL) system that enables efficient learning with minimal wet-lab data by integrating a molecular foundation model with an automated active-learning experimental workflow. Through ten iterative cycles, LUMI-lab synthesized and evaluated over 1,700 LNPs, identifying ionizable lipids with superior mRNA transfection potency in human bronchial cells compared to clinically approved benchmarks. Unexpectedly, it autonomously uncovered brominated lipid tails as a novel feature enhancing mRNA delivery. In vivo validation further confirmed that inhalation of LNPs containing the top-performing lipid, LUMI-6, achieved 20.3% gene editing efficacy in lung epithelial cells in murine models, surpassing the highest efficiency reported for inhaled LNP-mediated CRISPR-Cas9 delivery in mice to our knowledge. These findings demonstrate LUMI-lab as a powerful, data-efficient platform for advancing mRNA delivery, highlighting the potential of AI-driven autonomous systems to accelerate innovation in material science and therapeutic discovery.

Organisms that overwinter in temperate climates may experience freezing and freezing-induced oxidative stress during winter. While many insect species can survive freezing, molecular tools such as RNA interference (RNAi) or CRISPR have not been used to understand the physiological mechanisms underlying freeze tolerance. The spring field cricket Gryllus veletis can survive freezing following a 6-week fall-like acclimation. We used RNAi of an antioxidant enzyme in G. veletis to test the hypothesis that minimizing oxidative stress is important for freeze tolerance. In fat body tissue, Catalase mRNA abundance and enzyme activity increased during the acclimation that induces freeze tolerance. Other tissues such as midgut and Malpighian tubules had more stable or lower Catalase expression and activity during acclimation. In unacclimated (freeze-intolerant) crickets, RNA interference (RNAi) effectively knocked down production of the Catalase mRNA and protein in fat body and midgut, but not Malpighian tubules. In acclimated (freeze-tolerant) crickets, RNAi efficacy was temperature-dependent, functioning well at warm (c. 22°C) but not cool (15°C or lower) temperatures. This highlights a challenge of using RNAi in cold-acclimated organisms, as they may need to be warmed up for RNAi to work, potentially affecting their stress physiology. Knockdown of Catalase via RNAi in acclimated crickets also had no effect on the ability of the crickets to survive a mild freeze treatment, suggesting that Catalase may not be necessary for freeze tolerance. Our study is the first to demonstrate that RNAi is possible in a freeze-tolerant insect, but further research is needed to examine whether other genes and antioxidant molecules are important in freeze tolerance of G. veletis . Highlights Catalase expression and activity are elevated in freeze-tolerant cricket fat body RNAi knocks down Catalase in fat body and midgut at a warm temperature (22°C) RNAi is not effective at a cool temperature (15°C) that preserves freeze tolerance Catalase knockdown has no impact on survival of a mild freeze treatment The role of antioxidants in freeze tolerance warrants further study Graphical abstract

The impact of the genetic background on the lipidome of yeast strains remains underexplored. This study systematically compares the lipidomes of five commonly used laboratory yeast strains: BY4741, W303, D273-10B, RM11-1a, and CEN.PK2-1c. Shotgun lipidomics reveals significant variations in lipid class and acyl chain composition down to the level of molecular species. Notably, the most abundant lipid class differed between the strains: phosphatidylinositol (PI) lipids are predominant in BY4741, while phosphatidylethanolamine (PE) lipids are in D273. Ergosterol esters, which are the storage form of the major yeast sterol ergosterol, are at higher levels in all strains other than BY4741, correlating with a low gene expression of lipid metabolic enzymes Hmg1 and Are2 in BY4741. Despite these lipidomic differences, transcriptomic analysis did not show significant changes in most genes related to lipid metabolism, suggesting post-transcriptional modifications, protein abundance, and metabolic flux as potential regulatory mechanisms. This study underscores the complexity of lipidome regulation and the need for further investigation into the underlying mechanisms.

ABSTRACT Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with the SKI complex interacting exonuclease FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD -deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD -deleted cells. In vivo , HBS1L deletion eliminated growth of FOCAD -deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD -deleted tumors.

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

Telomere elongation is essential for the proliferation of cancer cells. Telomere length control is achieved by either the activation of the telomerase enzyme or the recombination-based Alternative Lengthening of Telomeres (ALT) pathway. ALT is active in about 10-15% of human cancers, but its molecular underpinnings remain poorly understood, preventing the discovery of potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable the unbiased discovery of molecular factors involved in cancer biology. Recently, Optical Pooled Screens (OPS) have significantly extended the capabilities of pooled functional genomics screens to enable sensitive imaging-based readouts at the single cell level and large scale. To gain a better understanding of the ALT pathway, we developed a novel OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout to measure ALT activity. The assay uses standard OPS protocols for library preparation and sequencing. As a critical element, an optimized nFISH protocol is performed before in situ sequencing to maximize the assay performance. We show that the modified nFISH protocol faithfully detects changes in ALT activity upon CRISPR knock-out (KO) of the FANCM and BLM genes which were previously implicated in ALT. Overall, the OPS-nFISH assay is a reliable method that can provide deep insights into the ALT pathway in a high-throughput format.

ABSTRACT Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALEN’s and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3.

ABSTRACT Pore-forming toxins (PFTs) are secreted bacterial effector molecules that disrupt host cell membranes. The α-hemolysin (HlyA) of uropathogenic Escherichia coli (UPEC) can exert damage to various mammalian cell types. While a candidate toxin receptor (CD11a/CD18 [LFA-1] integrin) exists on myeloid cells, the mechanism of HlyA cytotoxicity to epithelial cells remains undefined. We show that HlyA secretion by UPEC exacerbates renal tubular epithelial injury during ascending pyelonephritis in mice. A CRISPR-Cas9 loss-of-function screen in renal collecting duct cells identified clathrin-mediated endocytosis as required for HlyA cytotoxicity. HlyA internalization induces lysosomal permeabilization, facilitating protease release, cytoplasmic acidification, and mitochondrial dysfunction leading to rapid cell death. This mechanism contrasts with the described actions of other PFTs (plasma membrane poration and osmotic cytolysis). We also identify the low-density lipoprotein receptor (LDLR) as an epithelial receptor for HlyA; genetic or competitive inhibition of the HlyA-LDLR interaction prevented cytotoxicity. Our studies define a new mechanism of action for HlyA, in which its toxicity to epithelial cells requires LDLR-mediated, clathrin-dependent internalization. These results suggest therapeutic avenues for mitigating HlyA-induced damage during E. coli infections.

Streptococcus pneumoniae is an opportunistic pathogen responsible for life-threatening diseases including pneumonia and meningitis. The host defense against pneumococci relies heavily on macrophages, which can effectively internalize and degrade bacteria. Recent studies have implicated both canonical and non-canonical autophagy-related processes in bacterial clearance, but the precise pathways mediating defense against S. pneumoniae remain unknown. Here, we utilize a well-established zebrafish larval infection model to investigate the role of autophagy in host defense against pneumococci in vivo . Using a transgenic autophagy reporter line, we found the autophagy marker Lc3 being recruited to pneumococci-containing vesicles upon bacterial internalization by zebrafish macrophages. The genetic inhibition of core autophagy genes ( atg5 and atg16l1 ) led to loss of the Lc3 associations and their impaired acidification, significantly delaying bacterial clearance. This Lc3 recruitment is partially mediated by LC3-associated phagocytosis (LAP), as knockdown of cyba and rubcn moderately reduced Lc3 association with phagosomes and diminished pneumococcal degradation. Interestingly, we observed no involvement of xenophagy components in S. pneumoniae -infected macrophages, suggesting the activation of another non-canonical autophagy pathway, distinct from LAP, targeting pneumococci-containing phagosomes. Instead, we found that production of pneumococcal pore-forming toxin - pneumolysin induces LAP-independent Lc3 lipidation, which could be abolished by knockdown of tecpr1a indicating the involvement of the sphingomyelin-TECPR1-induced LC3 lipidation (STIL) pathway. Collectively, our observations shed new light on the host immune response against S. pneumoniae , demonstrating that two distinct non-canonical autophagy pathways mediate bacterial degradation by macrophages and providing potential targets for the development of novel therapies to combat pneumococcal infections.

Summary CHD3 is a component of the NuRD chromatin remodeling complex. Pathogenic CHD3 variants cause Snijders Blok-Campeau Syndrome, a neurodevelopmental disorder with variable features including developmental delays, intellectual disability, speech/language difficulties, and craniofacial anomalies. To unveil the role of CHD3 in craniofacial development, we differentiated CHD3 -KO induced pluripotent stem cells into cranial neural crest cells (CNCCs). CHD3 expression is low in wild-type iPSCs and neuroectoderm, but upregulated during CNCC specification, where it opens the chromatin at BMP-responsive enhancers, to allow binding of DLX5 and other factors. CHD3 loss leads to repression of BMP target genes and an imbalance between BMP and Wnt signalling, ultimately resulting in aberrant mesodermal fate. Consequently, CNCC specification fails, replaced by early-mesoderm identity, which can be partially rescued by titrating Wnt levels. Our findings highlight a novel role for CHD3 as a pivotal regulator of BMP signalling, essential for proper neural crest specification and craniofacial development.

Mitochondria control cellular metabolism, serve as hubs for signaling and organelle communication, and are important for the health and survival of cells. _VPS13D_ encodes a cytoplasmic lipid transfer protein that regulates mitochondrial morphology, mitochondria and endoplasmic reticulum (ER) contact, quality control of mitochondria. _VPS13D_ mutations have been reported in patients displaying ataxic and spastic gait disorders with variable age of onset. Here we used CRISPR/Cas9 gene editing to create _VPS13D_ related-spinocerebellar ataxia-4 (SCAR4) missense mutations and C-terminal deletion in _VPS13D_’s orthologue _vps-13D_ in _C. elegans_. Consistent with SCAR4 patient movement disorders and mitochondrial dysfunction, _vps-13D_ mutant worms exhibit locomotion defects and abnormal mitochondrial morphology. Importantly, animals with a _vps-13D_ deletion or a N3017I missense mutation exhibited an increase in mitochondrial unfolded protein response (UPRmt). The cellular and behavioral changes caused by _VPS13D_ mutations in _C. elegans_ advance the development of animal models that are needed to study SCAR4 pathogenesis.

ABSTRACT LMNA -associated congenital muscular dystrophy is a currently incurable rare genetic disorder characterized by early-onset muscle weakness, dilated cardiomyopathy and respiratory failure, resulting from mutations in the LMNA gene. In this study, we assessed the potential of a CRISPR-mediated strategy to eliminate the mutant allele Lmna c.745C>T, p.R249W using a mutation specific guide (sg745T). Results from R249W-mutation-carrying cellular models showed specific activity of the Cas9/sg745T complex towards the mutant allele. This property varied depending on the concentration of CRISPR components, with a loss of specificity observed with increased dosage. We tested this strategy in vivo using adeno-associated virus delivery in Lmna R249W mice. Despite being associated with a modest CRISPR activity, this therapeutic approach resulted in a 10% increase in the survival of R249W homozygous mice. Interestingly, a similar CRISPR activity improved the cardiac pathology developed by Lmna +/R249W animals, significantly extending their median survival. These results represent the first therapeutic validation of a CRISPR/Cas9-mediated gene editing strategy for the treatment of LMNA -associated congenital muscular dystrophy.

T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.

ABSTRACT Existing CRISPR-based genome editing tools are limited in Bacillus subtilis due to the large cas gene. The recently reported DNA nuclease IscB has the potential to be developed into a novel genome editing tool due to its size being one-third of Cas9, while its application in B. subtilis remains unexplored. In this study, genome editing tools pBsuIscB/pBsuenIscB based on IscB and enIscB (enhanced IscB) were established in B. subtilis SCK6, and successfully deleted 0.6 kb to 4.3 kb genes with efficiencies up to 100%. Subsequently, the pBsuenIscB with higher deletion efficiency was used, whereby the large genomic fragment of 37.7 kb or 169.9 kb was deleted with only one ωRNA. Additionally, single-copy or multi-copy mCherry genes was integrated by using pBsuenIscB. Finally, the editing plasmid was eliminated and the second round of genome editing was completed. Overall, this study has successfully applied IscB to B.subtilis , expanded the genome editing toolbox of B. subtilis , and will help to construct B. subtilis chassis for production of a variety of biomolecules.

Abstract OsbZIP35 is a member of the B subfamily of bZIP transcription factors in rice ( Oryza sativa L.). The function of OsbZIP35 has not been reported previously. In this study, drought, H 2 O 2 , abscisic acid, and NaCl treatments strongly induced the expression of OsbZIP35 , whereas treatment with gibberellin, indoleacetic acid, and jasmonic acid did not affect OsbZIP35 expression. We used the CRISPR/Cas9 gene-editing technology to construct OsbZIP35 knockout mutants ( bzip35-1 and bzip35-2 ) and evaluated the function of OsbZIP35 . The results showed that, compared with the wild type, the bzip35 mutants were more sensitive to drought stress during the germination, post-germination growth, and seedling stages. Specifically, the bzip35 mutants exhibited a lower germination percentage, a weaker growth phenotype, and an increase in reactive oxygen species accumulation. Further analysis indicated that, under drought stress, OsbZIP35 regulated reactive oxygen species accumulation by modulating the contents of antioxidants, thereby positively regulating the response of rice seedlings to drought stress. In addition, we observed that OsbZIP35 could be phosphorylated by OsSAPK3 and participated in the abscisic acid signaling pathway in response to drought stress. Agronomic trait analysis revealed that, under drought stress at the heading stage, the panicle length and seed-set rate of bzip35 mutants were significantly lower than those of the wild type. This study examined the role of OsbZIP35 in rice stress tolerance and yield regulation, and identified an upstream regulatory gene, OsSAPK3 . The results provide novel information on the mechanisms of stress tolerance and yield regulation in rice.

Our perception of the world depends on the brain’s ability to integrate information from multiple senses, with temporal disparities providing a critical cue for binding or segregating cross-modal signals 1,2 . The superior colliculus (SC) is a key site for integrating sensory modalities, but how cellular and network mechanisms in distinct anatomical regions within the SC contribute to multisensory integration remains poorly understood. Here, we recorded responses from over 5,000 neurons across the SC’s anatomical axes of awake mice during presentations of spatially coincident audiovisual stimuli with varying temporal asynchronies. Our findings revealed that multisensory neurons reliably encoded audiovisual delays and exhibited nonlinear summation of auditory and visual inputs, with nonlinearities being more pronounced when visual stimuli preceded auditory stimuli, consistent with the natural statistics of light and sound propagation. Nonlinear summation was crucial for population-level decoding accuracy and precision of AV delay representation. Moreover, enhanced population decoding of audiovisual delays in the posterior-medial SC, facilitated temporal discriminability in the peripheral visual field. Cross-correlation analysis indicated higher connectivity in the medial SC and functional specific recurrent connectivity, with visual, auditory, and multisensory neurons preferentially connecting to other neurons of the same functional subclass, and multisensory neurons receiving approximately 50 percent of the total local input from other multisensory neurons. Our results highlight the interplay between single-neuron computations, network connectivity, and population coding in the SC, where nonlinear integration, distributed representations and regional functional specialisations enables robust sensory binding and supports the accurate encoding of temporal multisensory information. Our study provides new insights into how the brain leverages both single-neuron and network-level mechanisms to represent sensory features by adapting to the statistics of the natural world.

The ability to identify gene functions and interactions in specific cellular contexts has been greatly enabled by functional genomics technologies. CRISPR-based genetic screens have proven invaluable in elucidating gene function in mammalian cells. Single-cell functional genomics methods, such as Perturb-seq and Spear-ATAC, have made it possible to achieve high-throughput mapping of the functional effects of gene perturbations by profiling transcriptomes and DNA accessibility, respectively. Combining single-cell chromatin accessibility and transcriptomic data via multiomic approaches has facilitated the discovery of novel cis and gene regulatory interactions. However, pseudobulk readouts from cell populations can often cloud the interpretation of results due to a heterogeneous response from cells receiving the same genetic perturbation, which could be mitigated by using transcriptional profiles of single cells to subset the ATAC-seq data. Existing methods to capture CRISPR guide RNAs to simultaneously assess the impact of genetic perturbations on RNA and ATAC profiles require either cloning of gRNA libraries in specialized vectors or implementing complex protocols with multiple rounds of barcoding. Here, we introduce CAT-ATAC, a technique that adds CRISPR gRNA capture to the existing 10X Genomics Multiome assay, generating paired transcriptome, chromatin accessibility and perturbation identity data from the same individual cells. We demonstrate up to 77% guide capture efficiency for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we were able to identify a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss of function experiments, we further validated that the gene, ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal novel gene and cellular interactions and functions.

Genome editing enzymes can introduce targeted changes to the DNA in living cells 1–4 , transforming biological research and enabling the first approved gene editing therapy for sickle cell disease 5 . However, their genome-wide activity can be altered by genetic variation at on- or off-target sites 6–8 , potentially impacting both their precision and therapeutic safety. Due to a lack of scalable methods to measure genome-wide editing activity in cells from large populations and diverse target libraries, the frequency and extent of these variant effects on editing remains unknown. Here, we present the first population-scale study of how genetic variation affects the cellular genome-wide activity of CRISPR-Cas9, enabled by a novel, sensitive, and unbiased cellular assay, GUIDE-seq-2 with improved scalability and accuracy compared to the original broadly adopted method 9 . Analyzing Cas9 genome-wide activity at 1,115 on- and off-target sites across six guide RNAs in cells from 95 individuals spanning four genetically diverse populations, we found that variants frequently overlap off-target sites, with 13% significantly altering Cas9 editing activity by up to 33% indels. To understand common features of high-impact variants, we developed a new massively parallel biochemical assay, CHANGE-seq-R, to measure Cas9 activity across millions of mismatched target sites, and trained a deep neural network model, CHANGE-net, to accurately predict and interpret the effects of single-nucleotide variants on off-targets with up to six mismatches. Taken together, our findings illuminate a path to account for genetic variation when designing genome editing strategies for research and therapeutics.

Mismatch repair (MMR) is a crucial DNA repair pathway that maintains genomic integrity by correcting replication errors and various forms of DNA damage. MMR deficiency (MMRd) leads to increased mutation rates, microsatellite instability, and contributes to tumorigenesis in multiple cancer types. Using a CRISPR-Cas9-mediated knockout assay in human isogenic cell lines, we characterised mutational profiles in MMR-deficient cells. Our findings revealed expected increases in mutation burden and the emergence of known MMR-associated mutational signatures. Notably, we identified a previously unconnected process, SBS57, and linked it to germline single-nucleotide polymorphisms and MMR-driven indels in MMRd cells, establishing its association with tensor signature TS27. Comparative analyses of in vitro MMRd profiles and in vivo tumour data uncovered key differences in mutational signatures, highlighting the biological context dependence of MMR-associated mutations. Furthermore, we provide direct experimental evidence that MMR plays a role in repairing 5-methylcytosine deamination, a repair process previously inferred from tumour sequencing data. These findings offer novel insights into MMR deficiency, shedding light on previously uncharacterised mutational mechanisms and their implications in cancer.

Photosynthesis is a biological process that converts light energy into chemical energy. Excessive light can damage the photosynthetic machinery, so plants have evolved photoprotective mechanisms such as non-photochemical quenching (NPQ). Among the NPQ mechanisms, qH is a form of sustained quenching, dependent on LIPOCALIN IN THE PLASTID (LCNP) and repressed by SUPPRESSOR OF QUENCHING 1 (SOQ1), protecting against abiotic stress. Recently, we showed in Arabidopsis thaliana that qH can occur in the major light-harvesting complexes (Lhcb1, Lhcb2, Lhcb3) but independently of any specific major antenna. Interestingly, in mutants with little or no accumulation of major antennae ( koLHCII, lhcb1, cpsrp43 ), qH can still be induced. Here, we show that the minor antennae can be quenched by qH and remain quenched once isolated. To investigate the role of minor antennae in qH, we combined the soq1 mutant, which displays high qH, with mutations in each minor antenna type (Lhcb4, Lhcb5, or Lhcb6), or with a mutant lacking all minor antennae. None are strictly required for qH to occur. Still, the absence of Lhcb6 decreases qH induction likely due to an indirect effect from the slower electron transport rate and/or a different macro-organization of photosynthetic complexes in the thylakoids. Overall, this work demonstrates that the minor antennae are a secondary target for qH and could serve as an additional safety valve for photoprotective energy dissipation during prolonged stress.