ABSTRACT To gain insight into biological mechanisms that cause resistance to DNA damage, we performed parallel pooled genetic CRISPR-Cas9 screening for survival in high risk HNSCC subtypes. Surprisingly, and in addition to ATM, DNAPK, and NFKB signaling, JAK1 was identified as a driver of tumor cell radiosensitivity. Knockout of JAK1 in HNSCC increases cell survival by enhancing the DNA damage-induced G2 arrest, and both knockout and JAK1 inhibition with abrocitinib prevent subsequent formation of radiation-induced micronuclei. Loss of JAK1 function does not affect canonical CDK1 signaling but does reduce activation of PLK1 and AURKA, kinases that regulate both G2 and M phase progression. Correspondingly, JAK1 KO was found to cause mitotic defects using both EdU labeling and live cell imaging techniques. Given this insight, we evaluated Kif18a inhibition as an approach to exacerbate mitotic stress and enhance the efficacy of radiation. These studies establish Kif18a inhibition as a novel strategy to counteract therapeutic resistance to DNA damage mediated by G2 cell cycle arrest.

Design: ing drugs that can restore a diseased cell to its healthy state is an emerging approach in systems pharmacology to address medical needs that conventional target-based drug discovery paradigms have failed to meet. Single-cell transcriptomics can comprehensively map the differences between diseased and healthy cellular states, making it a valuable technique for systems pharmacology. However, single-cell omics data is noisy, heterogeneous, scarce, and high-dimensional. As a result, no machine learning methods currently exist to use single-cell omics data to design new drug molecules. We have developed a new deep generative framework named MolGene-E to tackle this challenge. MolGene-E combines two novel models: 1) a cross-modal model that can harmonize and denoise chemical-perturbed bulk and single-cell transcriptomics data, and 2) a contrastive learning-based generative model that can generate new molecules based on the transcriptomics data. MolGene-E consistently outperforms baseline methods in generating high-quality, hit-like molecules from gene expression profiles obtained from single-cell datasets as validated by target knock-out experiments using CRISPR. This superior performance is demonstrated across diverse de novo molecule generation metrics. Extensive evaluations demonstrate that MolGene-E achieves state-of-the-art performance for zero-shot molecular generations. This makes MolGene-E a potentially powerful new tool for drug discovery.

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6–54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours ( e.g ., viral and non-viral vectors) or are not applicable in vivo ( e.g ., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LF N -Acr/PA, which delivers Acrs into cells within minutes. LF N -Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LF N -Acr binds and uses to enter the cell. In the presence of PA, LF N -Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LF N -Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LF N -Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

ABSTRACT Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo . Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins — from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 — while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system’s utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5′-exoribonuclease RNase J was identified as additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them PNPase. Despite a measured, strong 3’ exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase is a component of the cyanobacterial degradosome that can recruit either RNase E or RNase J, together with additional enriched proteins.

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5 - null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5 -null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

Summary Transitions between subsets of differentiating hematopoietic cells are widely regarded as unidirectional in vivo . Here, we introduce clonal phylogenetic tracer (CP-tracer) that sequentially introduces genetic barcodes, enabling high-resolution analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). This revealed previously uncharacterized HSC functional subsets and identified bidirectional fate transitions between myeloid-biased and lineage-balanced HSC. Contrary to the prevailing view that the more self-renewing My-HSCs unidirectionally transition to balanced-HSCs, phylogenetic tracing revealed durable lineage bidirectionality with the transition favoring My-HSC accumulation over time 1,2 . Further, balanced-HSCs mature through distinct intermediates—My-HSCs and lymphoid-biased-HSCs—with lymphoid competence here shown by CRISPR/Cas9 screening to be dependent on the homeobox gene, Hhex . Hhex enables Ly-HSC differentiation, but its expression declines with age. These findings establish HSC plasticity and Hhex as a determinant of myeloid-lymphoid balance with each changing over time to favor the age-related myeloid bias of the elderly. Highlights Sequenctial introduction of DNA barcodes in vivo was developed to assess time dependent changes in cell fate. Clonal phylogenetic tracer (CP-tracer) enabled high-resolution phylogenetic analysis of ∼100,000 subclones derived from ∼500 individual hematopoietic stem cells (HSC). Bidirectional fate transitions between myeloid-biased haematopoietic stem cells (My-HSCs) and lineage-balanced haematopoietic stem cells (balanced-HSCs) were observed. Hhex was identified as a molecular driver of HSC lymphoid competence.

ABSTRACT The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) regulates cell differentiation, proliferation, and function in various tissues, including the liver, adipose tissue, skin, lung, and hematopoietic system. Studies in rats, mice, humans, and chickens have shown that CEBPA mRNA undergoes alternative translation initiation, producing three C/EBPα protein isoforms. Two of these isoforms act as full-length transcription factors with N-terminal transactivation domains and a C-terminal dimerization and DNA-binding domains. The third isoform is an N-terminally truncated variant, translated from a downstream AUG codon. It competes with full-length isoforms for DNA binding, thereby antagonizing their activity. Expression of the truncated C/EBPα isoform depends on the initial translation of a short upstream open reading frame (uORF) in CEBPA mRNA and subsequent re-initiation at a downstream AUG codon, a process stimulated by mTORC1 signaling. We investigated whether the ortholog of the CEBPA gene in the evolutionarily distant, short-lived African turquoise killifish ( Nothobranchius furzeri ) is regulated by similar mechanisms. Our findings reveal that the uORF- mediated regulation of C/EBPα isoform expression is conserved in killifish. Disruption of the uORF selectively eliminates the truncated isoform, leading to unrestrained activity of the full-length C/EBPα isoforms. This genetic modification significantly extended both the median and maximal lifespan and improved the healthspan of male N. furzeri . These results highlight a conserved mechanism of CEBPA gene regulation across species and its potential role in modulating the lifespan and aging phenotypes.

SUMMARY Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans -Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the ‘druggable genome’, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans -Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

The chromatin regulator MLL2 (KMT2B) is the primary histone 3 lysine 4 (H3K4) trimethyltransferase acting at bivalent promoters in embryonic stem cells (ESCs) and is required for differentiation toward neuroectoderm. Here, we demonstrate that this requirement occurs during exit from naïve pluripotency, days before neuroectoderm differentiation is impaired. During exit, the effect of MLL2 on transcription is subtle, increasing the expression of a few important neuroectodermal transcription factors. In contrast, MLL2’s effect on chromatin architecture is substantial, stabilising loops associated with bivalent promoters in primed ESCs. MLL2 H3K4 catalytic activity is dispensable for stabilising these loops during ESC exit and for neuroectoderm differentiation. We therefore identify a non-catalytic function for MLL2 in stabilising 3D chromatin architecture, which has implications for lineage specification. Because MLL2 shares features with all four MLLs, we propose that chromatin tethering, rather than H3K4 methylation, represents a primary function for MLLs during lineage commitment decisions.

Mutations in the human SPTLC1 gene have recently been linked to early onset amyotrophic lateral sclerosis (ALS), characterized by global atrophy, motor impairments, and symptoms such as tongue fasciculations. All known ALS - linked SPTLC1 mutations cluster within exon 2 and a specific variant, c.58G>T, results in exon 2 skipping. However, it is unclear how the exon 2 deletion affects SPTLC1 function in vivo and contributes to ALS pathogenesis. Leveraging the high genomic sequence similarity between mouse and human SPTLC1 , we created a novel mouse model with a CRISPR/Cas9-mediated deletion of exon 2 in the endogenous murine Sptlc1 locus. While heterozygous mice did not develop motor defects or ALS-like neuropathology, homozygous mutants died prematurely. These findings indicate that Sptlc1 ΔExon2 heterozygous mice do not replicate the disease phenotype but provide valuable insights into SPTLC1 biology and serve as a useful resource for future mechanistic studies.

SUMMARY Extrachromosomal circular DNA (ecDNA) are commonly produced within the nucleus to drive genome dynamics and heterogeneity, enabling cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes mediate ecDNA production. Following DNA fragmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively catalyzes ecDNA production in Drosophila and mammals, with patient tumor ecDNA harboring junctions marked by LIG4 activity. Notably, disrupting LIG4 or BRCA1-A in cancer cells impairs ecDNA-mediated adaptation, hindering resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers new therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Oocyte cytoplasmic lattices are critical for early embryo development but their composition and function are not fully understood. Mutations in PADI6 , an essential component of cytoplasmic lattices, lead to early embryonic developmental arrest and female infertility. To investigate PADI6 function in mRNA storage, global protein levels, and lattice composition during early mammalian development we used single cell transcriptomics and proteomics methods to study two mouse models. Padi6 null mutation resulted in inhibition of embryonic genome activation, defective maternal mRNA degradation, and disruption to protein storage on the cytoplasmic lattices. Distinct developmental phenotypes were observed with a hypomorphic Padi6 mutation. By developing a powerful single cell proteomic fractionation method, we define the cytoplasmic lattice enriched proteome in which we find essential components of other major oocyte-specific compartments (ELVA, MARDO), suggesting previously unknown interconnections between them. Our findings highlight a critical scaffolding function of PADI6 and implicate cytoplasmic lattices as regulatory hubs for key processes in the oocyte and early embryo, including translation, respiration and protein degradation.

In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3’ biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.

Deep-sea hydrothermal vent ecosystems are sustained by chemoautotrophic bacteria that symbiotically provide organic matter to their animal hosts through the oxidation of chemical reductants in vent fluids. Hydrothermal vents also support unique viral communities that often exhibit high host-specificity and frequently integrate into host genomes as prophages; however, little is known about the role of viruses in influencing the chemosynthetic symbionts of vent foundation fauna. Here, we present a comprehensive examination of contemporary lysogenic and lytic bacteriophage infections, auxiliary metabolic genes, and CRISPR spacers associated with the intracellular bacterial endosymbionts of snails and mussels at hydrothermal vents in the Lau Basin (Tonga). Our investigation of contemporary phage infection among bacterial symbiont species and across distant vent locations indicated that each symbiont species interacts with different phage species across a large geographic range. However, our analysis of historical phage interactions via assessment of CRISPR spacer content suggested that phages may contribute to strain-level variation within a symbiont species. Surprisingly, prophages were absent from almost all symbiont genomes, suggesting that phage interactions with intracellular symbionts may differ from free-living microbes at vents. Altogether, these findings suggest that species-specific phages play a key role in regulating chemosynthetic symbionts via lytic infections, potentially shaping strain-level diversity and altering the composition and dynamics of symbiont populations.

Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce a tool kit for tagging genes by inserting artificial exons encoding fluorescent protein tags in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. So far, we have constructed 427 gRNA expression plasmids to target 178 genes as the first set. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. We provide a user-friendly database to organize the information of the cell line and pre-designed gRNAs at < https://yumahanaiatokamuralab.shinyapps.io/KnockInAtlas/ >.

Coronary artery disease (CAD) is the leading cause of death worldwide. Recently, hundreds of genomic loci have been shown to increase CAD risk, however, the molecular mechanisms underlying signals from CAD risk loci remain largely unclear. We sought to pinpoint the candidate causal coding and non-coding genes of CAD risk loci in a cell type-specific fashion. We integrated the latest statistics of CAD genetics from over one million individuals with epigenetic data from 45 relevant cell types to identify genes whose regulation is affected by CAD-associated single nucleotide variants (SNVs) via epigenetic mechanisms. Applying two statistical approaches, we identified 1,580 genes likely involved in CAD, about half of which have not been associated with the disease so far. Enrichment analysis and phenome-wide association studies linked the novel candidate genes to disease-specific pathways and CAD risk factors, corroborating their disease relevance. We showed that CAD-SNVs are enriched to regulate gene expression by affecting the binding of transcription factors (TFs) with cellular specificity. Of all the candidate genes, 23.5% represented non-coding RNAs (ncRNA), which likewise showed strong cell type specificity. We conducted a proof-of-concept biological validation for the novel CAD ncRNA gene IQCH-AS1 . CRISPR/Cas9-based gene knockout of IQCH-AS1 , in a human preadipocyte strain, resulted in reduced preadipocyte proliferation, less adipocyte lipid accumulation, and atherogenic cytokine profile. The cellular data are in line with the reduction of IQCH-AS1 in adipose tissues of CAD patients and the negative impact of risk alleles on its expression, suggesting IQCH-AS1 to be protective for CAD. Our study not only pinpoints CAD candidate genes in a cell type-specific fashion but also spotlights the roles of the understudied ncRNA genes in CAD genetics.

Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has become one of the most important gene editing tools. CRISPR guide RNA (gRNA) serves as an important component in guiding Cas proteins to a target site in gene editing processes. For effective gene editing or site specific gene integration, levels of gRNA present in a target cell are critical. Especially, for prime editing, even the ratio of different gRNAs present in target cells has a major effect on gene editing efficiency. Therefore, having a convenient, highly sensitive method for direct detection of gRNAs can improve editing design and optimize targeting efficiency. Here, we have developed a convenient, highly sensitive method for direct detection of gRNAs, which may be used to improve editing design and optimize targeting efficiency.

Trypanosoma cruzi, the causing agent of Chagas disease, is the only known trypanosomatid pathogenic to humans having a complete histidine to glutamate pathway, which involves a series of four enzymatic reactions that convert histidine into downstream metabolites, including urocanate, 4-imidazolone-5-propionate, N-formimino-L-glutamate and L-glutamate. Recent studies have highlighted the importance of this pathway in ATP production, redox balance, and the maintenance of cellular homeostasis in T. cruzi . In this work, we focus on the first step of the histidine degradation pathway, which is performed by the enzyme histidine ammonia lyase. Here we determined the kinetic and biochemical parameters of the T. cruzi histidine ammonia-lyase. By generating null mutants of this enzyme using CRISPR-Cas9 we observed that disruption of the first step of the histidine degradation pathway completely abolishes the capability of this parasite to metabolise histidine, compromising the use of this amino acid as an energy and carbon source. Additionally, we showed that the knockout of the histidine ammonia lyase affects metacyclogenesis when histidine is the only metabolizable source and diminishes trypomastigote infection in vitro .

Mycobacterium tuberculosis ( Mtb ) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb ( IS6110 and IS1081 ) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (10 5 to 10 2 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 – 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 – 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

Homeostasis relies on signaling networks controlled by cell membrane receptors. Although G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors, their specific roles in the epidermis are not fully understood. Dual CRISPR-Flow and single cell Perturb-seq knockout screens of all epidermal GPCRs were thus performed, uncovering an essential requirement for adhesion GPCR ADGRL2 (latrophilin 2) in epidermal differentiation. Among potential downstream guanine nucleotide-binding G proteins, ADGRL2 selectively activated Gα13. Perturb-seq of epidermal G proteins and follow-up tissue knockouts verified that Gα13 is also required for epidermal differentiation. A cryo-electron microscopy (cryo-EM) structure in lipid nanodiscs showed that ADGRL2 engages with Gα13 at multiple interfaces, including via a novel interaction between ADGRL2 intracellular loop 3 (ICL3) and a Gα13-specific QQQ glutamine triplet sequence in its GTPase domain. In situ gene mutation of this interface sequence impaired epidermal differentiation, highlighting an essential new role for an ADGRL2-Gα13 axis in epidermal differentiation.

Abstract The discovery of CRISPR-Cas systems and their antagonistic anti-CRISPR proteins (Acrs) exemplifies the perpetual arms race between bacteria and phages. While bacterial CRISPR-Cas systems function as adaptive immune mechanisms to combat phage infection, phages have evolved counterstrategies, such as various Acrs that disrupt CRISPR-mediated immunity via diverse molecular pathways. Here, we report the identification of a phage-encoded multi-protein system conferring anti-type I-F CRISPR activity. This system consists of three functionally distinct proteins: a RecA ATPase (SSAP), a DUF669 domain-containing protein (SSB), and a RecB exonuclease (Exo). Our mechanistic analysis reveals that SSB acts as a first-response mediator, forming a polymeric assembly to specifically binding to the Csy_dsDNA R-LOOP structure. This interaction creates a molecular platform enabling the coordinated recruitment of SSAP and Exo, which collectively execute homologous recombination-mediated repair of CRISPR-induced phage DNA break. These findings establish a paradigm of multi-protein synergy in phage counter-defense strategies, advancing our understanding of the intricate evolutionary dynamics in host-phage conflicts.

Advancements in biological and medical science are intricately linked to the biological central dogma. In recent years, gene editing techniques, especially CRISPR/Cas systems, have emerged as powerful tools for modifying genetic information, supplementing the central dogma and holding significant promise for disease diagnosis and treatment. Extensive research has been conducted on the continuously evolving CRISPR/Cas systems, particularly in relation to challenging diseases, such as cancer and HIV infection. Consequently, the integration of CRISPR/Cas-based techniques with contemporary medical approaches and therapies is anticipated to greatly enhance healthcare outcomes for human. This review begins with a brief overview of the discovery of the CRISPR/Cas system. Subsequently, using CRISPR/Cas9 as an example, a clear description of the classical molecular mechanism underlying the CRISPR/Cas system was given. Additionally, the development of the CRISPR/Cas system and its applications in gene therapy and high-sensitivity disease diagnosis were discussed. Furthermore, we address the prospects for clinical applications of CRISPR/Cas-based gene therapy, highlighting the ethical consideration associated with altering genetic information. This brief review aims to enhance understanding of the CRISPR/Cas macromolecular system and provide insight into the potential of genetic macromolecular drugs for therapeutic purposes.

ABSTRACT Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs in Europe. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster pathogenesis studies of visceral leishmaniasis. AUTHOR SUMMARY Visceral leishmaniasis is a disease caused by parasites of the Leishmania donovani complex. These parasites are spread to humans and animals through the bites of sand flies, and this disease affects millions of people worldwide, particularly in regions such as the Americas, Asia, Africa, and the Mediterranean basin. If left untreated, it can be fatal. Researchers need to study the biology of the parasite that causes the disease to better understand how it develops and progresses. In this study, we identified a L. infantum strain that is amenable to genetic modification in the laboratory and may serve as a representative model of the causative agent of visceral leishmaniasis. We tested two CRISPR-Cas9 strategies on this strain, re-sequenced and annotated its genome, and made the data available on the LeishGEdit website. By sharing this strain with the research community, we aim to support further studies on the pathogenesis of visceral leishmaniasis.

Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-simulated gene products, establishing a model for its broad antiviral activity. But, IFIT2 also bound viral mRNAs and enhanced their translation resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here we validate direct interactions between IFIT3 and RNA using electromobility shift assays (EMSAs). RNA-binding site identification (RBS-ID) experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability IFIT3 to promote IAV gene expression and translation efficiency when compared to wild type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers, however the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA-binding and its ability to modulate translation of host and viral mRNAs during IAV infection. Importance Influenza A viruses (IAV) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity can have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new work has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally as well as specifically in the context of IAV infection.