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.

Understanding how cells mitigate lysosomal damage is critical for unraveling pathogenic mechanisms of lysosome-related diseases. Here we use organelle-specific proteomics in iPSC-derived neurons (i 3 Neuron) and an in vitro lysosome-damaging assay to demonstrate that lysosome damage, caused by the aggregation of Ceroid Lipofuscinosis Neuronal 4 (CLN4)-linked DNAJC5 mutants on lysosomal membranes, serves as a critical pathogenic linchpin in CLN4-associated neurodegeneration. Intriguingly, in non-neuronal cells, a ubiquitin-dependent microautophagy mechanism downregulates CLN4 aggregates to counteract CLN4-associated lysotoxicity. Genome-wide CRISPR screens identify the ubiquitin ligase CHIP as a central microautophagy regulator that confers ubiquitin-dependent lysosome protection. Importantly, CHIP’s lysosome protection function is transferrable, as ectopic CHIP improves lysosomal function in CLN4 i 3 Neurons, and effectively alleviates lipofuscin accumulation and neurodegeneration in a Drosophila CLN4 disease model. Our study establishes CHIP-mediated microautophagy as a key organelle damage guardian that preserves lysosome integrity, offering new insights into therapeutic development for CLN4 and other lysosome-related neurodegenerative diseases.

Recently, there has been a significant outbreak of clinical pneumonia caused by the Human Metapneumovirus, particularly in Northern regions of the People’s Republic of China, causing thousands of hospitalisation cases. Such an outbreak has grown fresh concerns with regards to a potential spread of the novel infectious disease to several other world countries and causation of public health-related difficulties that may be similar in nature with the effects of the SARS-CoV-2-induced COVID-19 pandemic, which occurred from March 2020 to March 2022 before the disease finally became endemic in nature. Throughout the COVID-19 pandemic, a novel immunological research narrative was developed, in which a wider inclusion of natural immunity-based elements was recommended as part of an update in general approaches contained by immunotherapeutic and vaccine-related clinical research. Particularly, it has been suggested that a fairly decreased concentration of Type I and Type III elements from the host interferon system be placed in the central area of the natural immunity-based immunisation and immunisation adjuvance. Several clinical trials have confirmed the important position of such interferon system elements in the natural immunity department responsible for immunising functions. Given the fact that major components of the natural immune system have recently shown to display considerable adaptive immunity-like traits, such as specificity and long-term “memory”, natural immunity-based vaccination may now be deemed as scientifically plausible, contrary to initial scientific projections that they can only constitute vaccine adjuvants. Approaches as such may include a low dose of Type I and Type III Interferon-, and perhaps protollin-based treatment of nasopharyngeal tissues, as well as of natural and adaptive lymphocytes, and of plasmacytoid dendritic cells also, which represent both factories for Type I and Type III Interferons, as well as valid immune system-based vaccine candidates against infectious and oncological diseases, alongside natural and adaptive lymphocytes. Such components of the immune system may be utilised in combination to confer the most effective version of such an overall candidate of a clinical response. Other vaccine candidates may involve live-attenuated viral genomes either lacking the interferon-suppressive genes or containing them as the only slightly active genetic regions, with the overall purpose of stimulating an evolutionary push of the interferon-encoding genes to outcompete the already advanced stages of microbial evolution, whose stronghold seems to be largely upon the host interferon system. Other approaches may also involve the development of live-attenuated pathogen-derived vaccines that have Interferon I and III-encoding genes inserted into the viral genome as its sole active genetic components. There may be a novel, experimental process involving the isolation of common cold-inducing viruses, such as Rhinoviral agents, during the beginning of local, seasonal outbreaks and perhaps inducing their weakening as well, in clinical laboratories that are located in multiple distinct geographical areas of the hemisphere where the fall season has begun, prior to the performance of a small-scale gene editing through the insertion of active Type I and Type III Interferon-encoding genes into the genome of such viruses, prior to their release back into the local environment, via the performance of CRISPR-Cas9. Specifically, microbial genes involved in the causation of pathogenesis and maintenance of pathophysiology would be substantially attenuated, and genes involved in microbial reproduction and transmission perhaps not as much. Such a process may turn such viruses and possibly also other microbes into spreadable vaccines, as the immune system would automatically be activated once viruses as such undergo receptor-mediated endocytosis and start expressing their genes. At least some of the genes encoding proteins that antagonise the host interferon system could be removed prior to the insertion of human genes encoding various elements of it, particularly in situations where microbial agents are known to antagonise it in a problematic manner, whether directly or indirectly. If an overall procedure as such is performed accurately and matches all bioethical guidelines, then at least only common cold diseases in the upper respiratory tract, including the Rhinovirus-induced disease, may be prevented in many cases and probably even gradually become eradicated in the end, given the fact that an automatic synthesis of Type I and Type III Interferons by pathogenic microbes could lead to a robust and proportional rate of immune sensitisation that would lead to their lysis and disposal, making it probable that even microbes that are normally causative of major clinical disease would be destroyed before they would be able to induce the first symptoms. Active genes encoding Pattern Recognition Receptor (PRR) Activators matching to the microbe could also be inserted into its genome, perhaps to restore normative levels of microbial sensing by the host, natural immune system. Perhaps, inhalers and injectable sera containing a fairly decreased dosage of such potential transmissible factories for Type I and Type III Interferons, and possibly also for specific Pattern Recognition Receptor Activator and/or Agonist proteins, as such may be prepared to fill in any remote gap to the production of a full, herd-immunity effect throughout human populations. Such a potential overall update in vaccine innovation and development could even impact the evolutionary trajectory of various single drug and multidrug antibiotic resistant pathogenic bacteria, which can represent additional, unnecessary burdens for patients with various viral infections.

Neurons are highly asymmetric post-mitotic cells whose processes extend long distances to facilitate communication. Consequently, they encounter the complex problem of maintaining their structure and function over long distances. Over the last decade, research into the components composing the axon of a neuron has revealed the presence of local machinery of protein synthesis and deployment within different parts of the axon. However, there is still a need to understand how the endogenous proteome and transcriptome within the axon are distributed. The last decade has also witnessed a growth of technology capable of specifically labeling proteins and molecules of RNA. They are primarily based on gene editing techniques and recombinant antibody technology. Advances in technology now enable the delivery of large payloads of genetic material, paving the way for an in-depth investigation into the endogenous processes occurring within the axons of adult neurons. These capabilities open up exciting opportunities to address critical questions, potentially leading to new insights and strategies for treating neurodegenerative diseases. The review discusses different techniques available to a neuroscientist to help answer questions concerning the localization and transport of molecules within the axon. For instance, CRISPR is used to make specific changes to the genome and provide a means to tag endogenous proteins. Using these advances, in theory, it is possible to label molecules at scale and elucidate the role of different compartments that support protein synthesis and their subsequent deployment to specific regions within the axon.

Colorectal cancer (CRC) represents the third-leading cause of cancer-related deaths. Knowledge covering diverse cellular and molecular data from individual patients has become valuable for diagnosis, prognosis, and treatment selection. Here, we present an in-depth comparative mRNA-seq and microRNA-seq analysis of tissue samples from 32 CRC, pairing tumors with adjacent healthy tissues. The differential expression gene (DEG) analysis revealed an interconnection between nutrients, metabolic programs, and cell cycle pathways. We focused on the impact of overexpressed SLC7A11 (xCT) and SLC3A2 genes which compose the cystine/glutamate transporter (Xc-) system. To assess the oncogenic potency of the Xc-system in a cellular setting, we applied a knowledge-based approach for analyzing gene perturbations from CRISPR screens across various cell types as well as using a variety of functional assays in five primary patient-derived organoid cell models to functionally verify our hypothesis. We identified a previously undescribed cell surface protein signature predicting chemotherapy resistance and further highlighted the causality and potential of pharmacological blockage of ferroptosis as promising avenue for cancer therapy. Biological processes such as redox homeostasis, ion/amino acid transporters and de novo nucleotide synthesis were associated with these co-dependent genes in patient specimens. This study highlighted a number of overlooked genes as potential clinical targets for CRC and promotes stem cell-based, patient-individual in vitro model systems as a versatile partner platform to functionally validate in silico predictions, with focus on SLC7A11 and its associated genes in tumorigenesis.

Eukaryotic phototrophs depend on the activity of two engines (the plastid and the mitochondrion) to generate the energy required for cellular metabolism. Because of their overlapping functions, both activities must be closely coordinated. At the plastid level, optimization occurs through alternative electron transport, the diversion of excess electrons from the linear transport chain, and metabolic exchanges. A similar process takes place in the mitochondria, with documented evidence of energy and redox equivalents being exchanged between the two organelles. Organelle-organelle energy interactions at the physiological level are well established in diatoms, an ecologically significant member of phytoplankton. Yet the molecular components involved in this process remain largely unknown. Here, we identify a Mitochondrial Carrier Family (MCF) transporter, MCFc, located at the plastid envelope of Phaeodactylum tricornutum , which seems to be widely distributed in complex algae. We then compare the performance of a wild-type and a mutant lacking MCFc. An analysis of spectroscopic and oxygen exchange data unveiled altered energetic interactions in the mutant, suggesting that MCFc, plays a role in plastid-mitochondrion communication. In silico analysis of MCFc implies a similar substrate-specific model to that of ADP/ATP carriers, although distinct motif differences in MCFc indicate potential variations in its function, with possible substrates including arginine, aspartate/glutamate, or citrate. These findings illuminate how mitochondrial energy contributes to fueling diatom photosynthesis.

ABSTRACT We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans , a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with two separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans . This high-efficiency method requires two manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80 ). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology- dependent genome editing via selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans , including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans .

Artificial intelligence (AI), including machine learning (ML) and deep learning (DL), has become an essential tool in modern agriculture, revolutionizing traditional practices and offering sustainable solutions to critical challenges, such as climate change, population growth, and resource scarcity. Through advanced algorithms and predictive models, ML and DL enhance precise genomic selection (GS), trait characterization, and the acceleration of crop breeding processes. These technologies facilitate the identification and optimization of key traits, including increased yield, improved quality, pest resistance, and tolerance to extreme climatic conditions. Additionally, ML-driven tools support gene-editing technologies, such as CRISPR-Cas9, contributing to the development of resilient and adaptable crops. By leveraging big data analytics and omic technologies, they provide valuable insights into linking genetic and phenotypic data, fostering the development of sustainable agricultural practices. This research explores the transformative potential of AI, particularly ML and DL, in Solanaceous crops by developing advanced breeding strategies to address challenges posed by climate change and rapid population growth. Furthermore, this study highlights the significant role of these technologies in creating novel crop varieties that are resilient to environmental stressors, while exhibiting superior agronomic and quality traits. AI and its applications, such as ML and DL, contribute to the genetic improvement of Solanaceous crops, strengthening agricultural resilience, ensuring food security, and promoting environmental sustainability.

Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases could lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming, complex, and relatively costly. Here, we present CROFT-Seq ( CR ISPR nuclease of f- t arget detection by seq uencing), a sensitive, rapid, and cost-effective assay for the genome-wide detection of Cas9 off-target sites in vitro . CROFT-Seq performs comparably to the common currently used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-targets identified by CROFT-Seq were validated in cells, highlighting its effectiveness as a predictor of off-target sites.

Cisplatin chemotherapy of colorectal cancer (CRC) is associated with dose-limiting side effects and the development of drug resistance, resulting in reduced therapeutic effectiveness. The resistant phenotype in colon cancer is primarily due to changes in p53-regulated DNA damage signaling and /or defects in the cellular mismatch-repair pathway. Therefore, enhancing the efficacy of cisplatin chemotherapy remains a significant challenge. In this study, we used a TP53-KO patient-derived colon tumor organoid model to perform a genome-wide CRISPR KO screen in the absence and presence of cisplatin and identified gene knockouts that re-sensitize cisplatin-resistant TP53-KO colon cancer organoids to cisplatin treatment. Knockout of genes in the DNA Repair pathways, including Fanconi Anemia (FA cause re-sensitization of TP53-KO colon cancer cells to cisplatin. Inhibition of genes ERCC6, FANCL, and BRIP1 enhances cisplatin-induced cell death in TP53-KO colon cancer organoids. These findings suggest that targeting these pathways could be an effective approach to overcome chemoresistance of TP53-muatnt colon cancer cells to cisplatin.

Design: ing CRISPR single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we present ALLEGRO, a combinatorial optimization algorithm able to design minimal, yet highly effective sgRNA libraries targeting thousands of species. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting several genes of interest for over 2,000 species across the fungal kingdom. We experimentally validated the sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii , and Yarrowia lipolytica . In addition, we adopted a generalized Cas9-Ribonucleoprotein delivery system coupled with protoplast transformation to extend ALLEGRO’s sgRNA libraries to other untested fungal genomes, such as Rhodotorula araucariae . Our experimental results, along with cross-validation, show that ALLEGRO enables efficient CRISPR genome editing, supporting the development of universal sgRNA libraries applicable to entire taxonomic groups.