ABSTRACT The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability in vitro and in vivo and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

Abstract Background The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play an important role in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. Methods An RNA-seq analysis was used to identify genes differentially impacted by SR8278 treatment in human HaCaT keratinocytes, which was confirmed by RT-qPCR and western blotting. Cell growth and viability assays were further used to examine cell proliferation in HaCaT and other cell lines. CRISPR/Cas9 genome editing was used to generate cells lacking REV-ERBα and β. Results RNA-seq analysis indicated genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, which was confirmed via RT-qPCR and western blotting. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce apoptosis. Finally, the knockout of the REV-ERBs did not impact the effect of SR8278 on gene expression and cell proliferation. Conclusions We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and thus care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown.

Genome replication start sites, called origins, begin to be specified by Origin Recognition Complex (ORC) proteins prior to replication through a process called origin licensing. Once licensed, origins become active and initiate DNA synthesis with varying efficiencies influenced by local chromatin environment, transcription, and 3D genome organization. ORC proteins have also been implicated in regulating chromatin state and nuclear organization. However, it is unclear if there is interplay between ORC and the chromatin architectures underlying origin activation, as we lack a systems-level understanding of how ORC proteins interact, post-licensing, with the nuclear environments conducive to genome synthesis. To infer this context-specific ORC interactome, I used data from genome-wide CRISPR fitness screens, a cell-wide proximity labeling study, and proteomic profiling of nascent DNA-associated proteins to identify 17 novel factors that genetically and proteomically interact with ORC subunits and genome replication. Unexpectedly, the candidate pool was significantly enriched for factors involved in the homeostasis of RNA Polymerase III (Pol III) transcripts, particularly 5S ribosomal RNA (rRNA) and transfer RNA (tRNA). Follow-up protein-protein structure predictions by AlphaFold 3 (AF3) proposed direct interactions between ORC subunits and Pol III transcript biogenesis factors, as well as epigenetic regulators and a cyclin-dependent kinase. Given the prominence of 5S rRNA and tRNA biogenesis factors in my results, and prior reports of ORC subunits binding RNA, I also used protein-RNA structure prediction to identify candidate ORC3 interactions with 5S rRNA and tRNA. Altogether, my analysis integrates biological process, molecular proximity in human cells, and structural prediction to nominate novel protein and RNA interactions for involvement in the human replication program. These results augment and expand current models for ORC function and origin activation, particularly those involving chromatin state and transcriptional activity, and generate testable hypotheses to explore the interdependencies of replication patterning, histone modification, and nuclear RNA homeostasis.

Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis . In this method, the enzymatic activity of an inactive luciferase mutant , LUC40Ins26bp , which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp , which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.

ABSTRACT CRISPR-Cas9–mediated gene editing was used to generate specific mutants of the bantam gene in Drosophila melanogaster . To drive non-homologous end joining (NHEJ) and achieve a precise deletion of most of the bantam locus, two guide RNAs targeting sites 90 base pairs apart were expressed in the germline using the UAS/GAL4 system. Thirty lethal and eight viable lines were established and analyzed. One lethal line exhibited the expected 90 bp deletion, while the others carried diverse indels at one or both cleavage sites. Among the viable lines, seven harbored a single-nucleotide deletion that did not disrupt bantam function. Notably, one viable line, ban d1-44 , carried a hypomorphic allele that reduced organismal size without affecting viability. To generate precisely edited bantam variants, CRISPR-Cas9–mediated homology-directed repair (HDR) was used using donor plasmids containing engineered mutations in the miRNA seed region, along with a scarless dsRED fluorescent marker. Approximately 40% of the resulting fluorescent lines were correctly edited, demonstrating the efficiency of this strategy for producing specific bantam variants. The remaining lines exhibited unexpected outcomes, among which partial HDR events, where the dsRED marker was integrated but not the bantam mutations, and full donor plasmid integrations, which led to duplication of the bantam locus. These findings reveal the complexity of CRISPR-Cas9 outcomes, emphasizing the need for thorough screening and characterization of individual candidates in gene-editing experiments. They also provide valuable insights for optimizing genome editing strategies. Article summary The authors used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing to mutate the bantam microRNA in Drosophila melanogaster . They guided Non-Homologous End Joining to induce a precise 90 bp deletion. It was the rarest occurrence, while small inactivating indels occurred frequently. They employed Homology-Directed Repair using a fluorescent marker to specifically target the bantam seed region. This efficiently produced the intended mutations but also led to unexpected outcomes, including partial sequence replacements and full donor plasmid integrations. These results reveal the complexity of gene editing outcomes and highlight the importance of thorough molecular characterization in genome engineering experiments.

TBLR1 is a subunit of the NCoR corepressor complex that is mutated in a range of neurodevelopmental disorders. Here, we report that TBLR1 functions as a molecular scaffold that physically connects ANKRD11 and SETD5 – two of the most frequently mutated proteins in neurodevelopmental disorders – and links them to the rest of the NCoR complex. The resulting assembly resembles the yeast SET3 complex (SET3C) – a transcriptional regulator. Pathogenic missense mutations in TBLR1, ANKRD11 and SETD5 disrupt this assembly, and an engineered mutation that specifically abolishes SETD5 incorporation into SET3C causes severe developmental impairments in mice. Disruptions of mammalian SET3C components cause highly correlated changes in gene expression – including upregulation of already highly transcribed genes. Together, our results reveal that failure of transcriptional regulation by SET3C is a convergent molecular basis for a family of neurodevelopmental disorders.

Summary We report the development of REMEDY (REpair of heterozygous Mutations independent of Exogenous Donor template with high efficiencY), a genome editing strategy that allows efficient repair of heterozygous mutations in human and mouse cells without necessitating an exogenous donor DNA template. Here, we used in-PAM or near-PAM CRISPR strategies to induce a double-strand break (DSB) in mutant alleles. Following the DSB, the wild-type homologous chromosome itself serves as an endogenous DNA donor template and initiates the correction of the mutant allele. Concurrently treating the cells with HDR enhancers, such as AZD7648, further improved the efficiency of the correction. We demonstrated the utility of REMEDY in the context of six different diseases with heterozygous mutations such as IBMPFD, cystic fibrosis, progeria, ITPR3-associated combined immunodeficiency, ACTA1, and TBCD in human patient derived primary cells and complementary mouse model cell lines. Abstract Figure Graphical Abstract

Groundwater ecosystems harbor diverse microbial communities adapted to energy-limited, light-deprived conditions, yet the role of viruses in these environments remains poorly understood. Here, we analyzed 1.26 terabases of metagenomic and metatranscriptomic data from seven wells in the Hainich Critical Zone Exploratory (CZE) to characterize groundwater viromes. We identified 257,252 viral operational taxonomic units (vOTUs) (>=5 kb), with 99% classified as novel, highlighting extensive uncharacterized viral diversity. Viruses exhibited a distinct host range, primarily targeting Proteobacteria, Candidate Phyla Radiation (CPR) bacteria, and DPANN archaea. Notably, CPR lineages displayed low virus-host ratios and viral CRISPR targeting multiple hosts, suggesting a virus decoy mechanism where they may absorb viral pressure, protecting bacteria hosts. Additionally, 3,378 vOTUs encoded auxiliary metabolic genes (AMGs) linked to carbon, nitrogen, and sulfur cycling, with viruses targeting 31.5% of host metabolic modules. These findings demonstrate viruses influence on microbial metabolic reprogramming and nutrient cycling in groundwater, shaping subsurface biogeochemistry.

The increasing global prevalence of Mycobacterium abscessus infections presents a significant clinical challenge due to the pathogen’s intrinsic resistance to multiple antibiotics and poor treatment outcomes. Despite the necessity of genetic tools for studying its physiology, pathogenesis, and drug resistance, efficient methods for large-fragment deletions remain underdeveloped. Here, we report a CRISPR/Cas9-based dual-sgRNA system employing Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9), enabling efficient large-fragment knockout in M. abscessus with deletion efficiencies exceeding 90% at certain loci and spanning up to 16.7 kb. Furthermore, we systematically optimized the modular arrangement of genetic components in Cas9/dual-sgRNA expression plasmids and refined their construction workflow, achieving a significant reduction in cassette loss rates while enabling single-step plasmid assembly. Notably, deletion efficiency was position-dependent rather than correlated with target size, suggesting an influence of chromatin structure on editing outcomes. As the first CRISPR/Cas9-based platform capable of kilobase-scale deletions in M. abscessus , this system advances functional genomics studies and facilitates targeted investigations into virulence and antibiotic resistance mechanisms.

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin–streptavidin capture within 5 minutes. The CRISPR/Cas12a–tripod–LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

Host cells contest invasion by intracellular bacterial pathogens with multiple strategies that recognise and / or damage the bacterial surface. To identify novel host defence factors targeted to intracellular bacteria, we developed a versatile proximity biotinylation approach coupled to quantitative mass spectrometry that maps the host-bacterial interface during infection. Using this method, we discovered that intracellular Shigella and Salmonella become targeted by UFM1-protein ligase 1 (UFL1), an E3 ligase that catalyses the covalent attachment of Ubiquitin-fold modifier 1 (UFM1) to target substrates in a process called UFMylation. We show that Shigella antagonises UFMylation in a dual manner: first, using its lipopolysaccharide (LPS) to shield from UFL1 recruitment; second, preventing UFM1 decoration by the bacterial effector IpaH9.8. Absence of UFMylation leads to an increase of bacterial burden in both human cells and zebrafish larvae, suggesting that UFMylation is a highly conserved antibacterial pathway. Contrary to canonical ubiquitylation, the protective role of UFMylation is independent of autophagy. Altogether, our proximity mapping of the host-bacterial interface identifies UFMylation as an ancient antibacterial pathway and holds great promise to reveal other cell-autonomous immunity mechanisms.

ABSTRACT The fungal pathogen Candida albicans colonises the human gut where short-chain fatty acids (SCFAs) offer sources of carbon. This fungus harbours one of the largest microbial families of ATO (Acetate Transport Ortholog) genes, which encode putative SCFA transport proteins. Here, we generate C. albicans null mutants lacking individual or all known putative SCFA transporter genes and compare their phenotypes in vitro and in vivo . We show that blocking ATO function in C. albicans impairs SCFA uptake and growth, particularly on acetate. The uptake of acetate is largely dependent on a functional Ato1 (also known as Frp3/Ato3) and it is effectively abolished upon deletion of all ATO genes. We further demonstrate that deletion of the entire ATO gene family, but not inactivation of ATO1 alone, compromises the stable colonisation of C. albicans in the murine gastrointestinal tract following bacterial disruption by broad-spectrum antibiotics. Our data suggest that the ATO gene family has expanded and diversified during the evolution of C. albicans to promote the fitness of this fungal commensal during gut colonisation, in part through SCFA utilisation. IMPORTANCE The human gut is rich in microbial fermentation products such as SCFAs, which serve as key nutrients for both bacteria and fungi. C. albicans , a common fungal resident of the gut and a cause of opportunistic infections, carries an unusually large family of ATO genes. This study reveals that this ATO gene family is required for the efficient uptake of acetate, the most abundant SCFA in the gut, and for stable colonisation of the gut. These findings uncover a new layer of metabolic adaptation in fungal commensals of humans and suggest that transporter gene expansion can shape microbial fitness in response to environmental nutrient signals.

Abstract Aim ; The study was conducted to establish the association between CRISPR-Cas system in Pseudomonas aeruginosa clinical and environmental strains to the sensitivity to phages and antibiotics. Method; In this study, the occurrence and distribution of CRISPR-Cas system was analyzed across the 50 Pseudomonas aeruginosa strains. Further, to evaluate the role of CRISPR-Cas system as inhibitor of HGT, the role of CRISPR-Cas system in relation to susceptibility of Pseudomonas aeruginosa to various antibiotics and phages was studied. Result : A total of 5 different types of Type I CRISPR- Cas systems i.e. Type IF, Type IE, Type IC, Type IV and Type IU were identified in P. aeruginosa strains. A total of 5 (17%) clinical strains and 8 (40%) environmental strains were positive for both CRISPR and cas3 gene. The analysis of phage as well as antibiotic susceptibility of both clinical and environmental strains revealed 36% clinical strains and75% environmental strains were found to be resistant to phages. Antibiotic susceptibility results were in contrast to phage susceptibility as 50% clinical strains were resistant to at least 3 antibiotics i.e. they were multidrug resistant (MDR) while only 15% environmental strains were classified as MDR. Conclusion : The phage susceptibility and antibiotic resistance data was found to be correlating to presence of CRISPR-Ca system in the P. aeruginosa .

Resistance to androgen receptor (AR)-targeted therapies, such as enzalutamide, in castration-resistant prostate cancer (CRPC) remains a significant clinical challenge, often driven by mechanisms including lineage plasticity. The precise molecular mechanisms driving this process, particularly downstream effectors, remain incompletely understood. Given its established roles in cell fate and stemness, alongside its complex functions in prostate cancer, the Notch signaling pathway presented a compelling focus for study. This study investigates the role of Notch signaling in mediating lineage plasticity and therapeutic resistance in CRPC. Employing transcriptomic analysis and functional assays, we identified Notch activity is elevated across prostate cancer progression resistance. Notably, both CRISPR-mediated knockout and targeted inhibition of Notch reversed enzalutamide resistance in vitro . Collectively, this study delineates dynamic alterations in Notch signaling activity during prostate cancer progression and establishes its function as a crucial and druggable driver of therapy resistance. These findings underscore Notch signaling as a promising therapeutic target to counteract resistance to AR-targeted therapies in advanced prostate cancer.

Transmission of Plasmodium parasites to the Anopheles vector critically depends on swift activation of mature gametocytes upon entry into the mosquito midgut. Induction of gametogenesis requires two simultaneous stimuli, a temperature drop and xanthurenic acid. Previous work in the murine malaria model Plasmodium yoelii identified a protein, termed gametogenesis essential protein ( GEP1 ), with a suggested role in xanthurenic acid-dependent activation of gametes. Here, we present an experimental genetics characterization of GEP1 in the human pathogen Plasmodium falciparum . Using CRISPR-Cas9 gene editing we generated PfGEP1 loss-of-function lines and analyzed their progression until gametocyte maturation. We show a complete defect in both male and female gametogenesis caused by disruption of PfGEP1 . Pfgep1(-) gametocytes do not produce gametes when activated with xan-thurenic acid or a drop in temperature. This defect could not be overcome by the phosphodiesterase inhibitor Zaprinast, which induces gametogenesis. We also explored GEP1 haplotypes in P. falciparum parasites circulating in endemic regions and show the presence of two non-synonymous SNPs, resulting in V241L and S263P mutations, in 12% and 20% of 49 sentinel samples, respectively. Together, our data indicate that GEP1 plays a central role in the gamete activation process independent of xanthurenic acid and validates Pf GEP1 as a promising transmission blocking target.

We present a generalisable, interpretable machine learning framework for therapeutic target discovery using single-cell transcriptomics, protein interaction networks, and drug proximity analysis. The pipeline integrates feature selection via gradient boosting classifiers, systems-level network inference, and in silico drug repurposing, enabling the identification of actionable targets with cellular specificity. As a proof of concept, we apply the method to clear cell renal cell carcinoma (ccRCC), an aggressive kidney cancer with limited treatment options. The model identifies 96 tumour-intrinsic genes, refines them to 16 targets through CRISPR screens and biological curation, and prioritises FDA-approved compounds via network-based proximity scoring. Several novel therapeutic mechanisms - including ABL1, CDK4/6, and JAK inhibition - emerge from this analysis, with predicted compounds showing superior efficacy to standard-of-care drugs across multiple ccRCC cell lines. Beyond ccRCC, this framework offers a scalable strategy for drug discovery across diverse diseases, combining machine learning interpretability with systems biology to accelerate therapeutic development.

Advances in genome engineering and single-cell RNA sequencing (scRNAseq) have revolutionized the ability to precisely map gene functions, yet scaling these techniques for large-scale genetic screens in animals remains challenging. We combined high-throughput gene disruption in zebrafish embryos via Multiplexed Intermixed CRISPR Droplets with phenotyping by multiplexed scRNAseq (MIC-Drop-seq). In one MIC-Drop-seq experiment, we intermixed and injected droplets targeting 50 transcriptional regulators into 1,000 zebrafish embryos, followed by pooled scRNAseq. Tissue-specific gene expression and cell abundance analysis of demultiplexed mutant cells recapitulated many known phenotypes, while also uncovering novel functions in brain and mesoderm development. We observed pervasive cell-extrinsic effects among these phenotypes, highlighting how whole-embryo sequencing captures complex developmental interactions. Thus, MIC-Drop-seq provides a powerful and scalable platform for mapping gene functions in vertebrate development with cellular resolution.

The penetrant PRKCA D463H mutation, a biomarker and potential driver in chordoid glioma, was found to provoke the development of chondrosarcomas in heterozygous knock-in mice. This mutation entirely abrogates kinase activity, but strikingly no oncogenic phenotype is observed for the related inactivating mutation D463N indicating that the lack of activity is not the driver. In cells, the D463H mutant closely mirrored PKCα WT behaviours and retained ATP binding, contrary to the related D463N mutant. Mechanistically, the PKCα D463H mutant protein was found to display quantitative alterations to the PKCα interactome, enhancing association with epigenetic regulators. This aligned with transcriptomic changes which resembled an augmented PKCα expression program, with enhanced BRD4, Myc and TGFβ signatures. D463H dependent reduced sensitivity to the BET inhibitors JQ1 and AZD5153 indicates the functional importance of these pathways. The data show that D463H is a dominant gain-of-function oncogenic mutant, operating through a non-catalytic allosteric mechanism. One Sentence Summary A PKCα catalytic inactivating mutation confers gain-of-function properties - a paradigm shift in kinase actions.

Abstract Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract. In recent years, secondary resistance to the first-line drug imatinib has become its bottleneck of targeted therapy due to the unclear mechanism. It has important clinical significance for breaking through the bottleneck by screening and identifying the critical gene of imatinib resistance. Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms. Here the genome-scale CRISPR/Cas9 Knockout Screening was applied to investigate imatinib resistance genes in GIST 882 cell line for two rounds, and it was found that deficiency of sphingosine 1-phosphate lyase coding gene SGPL1 can inhibit tumor cell apoptosis and accelerate cell cycle G1/S, finally leading to imatinib resistance in vitro and in vivo, by regulating the expression of Bcl-2, p27kip1 and p15INK4B via PI 3 K-Akt signaling pathway. In additionally, non-synonymous mutation in the exon of SGPL1 gene has been found by comparing the TCGA clinical drug resistance patient database. It was revealed that SPGL1 gene may be the critical gene of imatinib resistance. Taken together, our study provides a resource for achieving a deep understanding of the molecular basis of imatinib resistance.

Abstract CRISPR/Cas9 technology is an efficient tool for livestock gene editing. However, host genome function can be disrupted by the random integration of exogenous genes. To circumvent this issue, site-specific integration is required. This study established a multi-dimensional assessment system to evaluate the biological applicability of H11/Rosa26 safe harbor loci as targeted integration platforms for exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study provides the first demonstration of H11/Rosa26 loci as dual-functional sites that enable both high-efficiency integration and biosafety in goat models using a cross-scale (cellular-embryonic-individual) validation system, offering a precise and low-risk technical paradigm for livestock genetic improvement.

ABSTRACT Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus as in CRISPR-Cas adaptive immunity systems and horizontal gene transfer (HGT) which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate simultaneously, but empirical observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history tradeoffs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable and fluctuating, unpredictable environments with a moderate viral prevalence, direct interaction with the virus and horizontal transfer of defense genes become the optimal routes of immunity acquisition, respectively. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles. IMPORTANCE The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via horizontal gene transfer (HGT) whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short term evolution of prokaryotic immunity and show that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.

The molecular mechanisms that enable memories to persist over long time-scales from days to weeks and months are still poorly understood. To develop insights we created a behavioral task where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. We then monitored circuit-specific molecular programs that diverge between consolidated and forgotten memories. We identified multiple distinct waves of transcription, i.e., cellular macrostates, specifically in the thalamo-cortical circuit, that defined memory persistence. Notably, a small set of transcriptional regulators orchestrated broad molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent, causal, and strikingly time-dependent roles in memory stabilization. In particular, the calmodulin-dependent transcription factor Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase Ash1l were required later to maintain memory over weeks. These results identify a critical Camta1-Tcf4-Ash1l thalamo-cortical transcriptional cascade required for memory stabilization, and puts forth a model where the sequential, multi-step, recruitment of circuit-specific transcriptional programs enable memory maintenance over progressively longer time-scales.

ABSTRACT The placenta is an important producer of hormones essential for fetal development. Insulin-like growth factor 1 (IGF1) is a hormone primarily produced in the placenta in utero and is an important regulator of various developmental pathways including those in heart and liver. Embryonic disruptions in these developmental pathways can lead to lifelong changes and are often associated with chronic disease. Further, the placenta has sex-specific impacts on offspring development in response to hormonal changes. Previous work has shown that altered expression of Igf1 in the placenta results in sexually dimorphic changes to placental and fetal developmental outcomes. Here, mice underwent placental-targeted CRISPR manipulation for overexpression or partial knockout of Igf1 . At the time of euthanasia, heart and liver tissues were collected and weighed. This dataset presents the heart and liver mass of these postnatal mice. There was a significant increase in proportional heart mass in placental Igf1 overexpression adult female mice and a trending increase in proportional liver mass in placental Igf1 overexpression adult male mice. No significant changes in heart or liver mass were seen in placental Igf1 partial knockout mice. These data provide insight into the impact of placental IGF1 on long-term heart and liver development. VALUE OF THE DATA There is significant evidence for the role of early genetic changes in influencing long-term health outcomes, as laid out by the Developmental Origins of Health and Disease (DOHaD) hypothesis [1]. According to this hypothesis, genetic factors may be critical in determining the timing and severity of chronic disease, with varying effects based on sex. Genetics of the placenta, which makes up the maternal-fetal interface, plays an important role in modulating exposures associated with the DOHaD hypothesis [2]. The placenta provides essential hormones to the fetus during pregnancy [3]. Placental changes are associated with the development of chronic disease and metabolic changes [4,5]. Disruptions in placental functions have been linked to defects including congenital heart disease which affects approximately 40,000 babies each year in the United States [6,7]. The placenta is also linked to metabolic diseases later in life such as nonalcoholic fatty liver disease, a chronic liver disease which has increased in prevalence by over 50% from 1990 to 2019 [5,8,9]. Insulin-like growth factor 1 (IGF1) is a placentally produced factor that regulates pathways involved in fetal growth and development and has been shown to be critical in growth of the heart and liver [10-13]. Despite the importance of the placenta and IGF1 in heart and liver growth, specific links between placental Igf1 expression and developmental outcomes remain understudied. Placental function is known to have sex-specific impacts on fetal growth [14]. Further, Igf1 expression in the placenta is linked to differences in offspring developmental outcomes by sex [15]. Placental Igf1 overexpression and knockout affects offspring in a sexually dimorphic manner. IGF1 is a hormone and interacts with sex hormones, likely contributing to sex differences in response to changes in Igf1 expression [16]. Further research, including the work done to produce this dataset, may help clarify the role of placenta Igf1 expression in fetal outcomes, specifically regarding sex differences. The data presented in this paper provide insight into the effects of placental Insulin-like growth factor 1 overexpression and partial knockout on adult heart and liver mass. More research is needed to understand specific functional impacts on these organs. Further, understanding the effects of placental genetic changes may support the development of future treatments and therapies for placental insufficiencies.

Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH 4 ) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a methanotroph CRISPR interference (CRISPRi) system to enable functional genomic screening. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus , consisting of ∼45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH 4 identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs, including the identification of novel essential genes. M. capsulatus growth was inhibited when the CRISPRi system was used to individually target genes identified in the screen, validating their essentiality for methanotrophic growth. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy. These CRISPRi screening methodologies can also be applied to high-throughput engineering approaches for isolation of improved methanotroph biocatalysts.

Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a cutting-edge workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly precursors corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins with various TPD approaches. Therefore, our workflow holds tremendous potential for rapidly establishing mode of action for various TPD modalities, including PROTACs and molecular glues.