CRISPR/Cas mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus ( Vpa CASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the Vpa CASTm and their associated CRISPR arrays ( Vpa CAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the Vpa CAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus . Additionally, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO and H-NS. Since it was previously reported that the activity of the Vpa CAST system was less efficient than that of the Vch CAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli , we analyzed if the transposition efficiency mediated by the Vpa CAST system could be enhanced inside its natural host V. parahaemolyticus . We provide evidence that this might be the case suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs. Importance Mobile genetic elements such as transposons play important roles on the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli .

Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated omic and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit growth with inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated, “druggable” targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.

Abstract Leaf rust is a devastating fungal disease of wheat. Planting resistant wheat cultivars is the most effective strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr30 / Lr.ace-4A encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem NB-ARC domains. This gene proves both necessary and sufficient to confer resistance to Puccinia triticina , as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr30 provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr30 haplotypes, with transgenic plants carrying either variant exhibiting susceptibility. Cloning of Lr30 will accelerate its deployment in wheat breeding programs.

Lack of canonical biomarkers and strategies to target radioresistance contribute to poor patient outcomes in triple-negative breast cancer (TNBC). Identifying and targeting novel radioresistance genes will benefit in enhancing radiotherapy response and treatment outcome in TNBC patients. A genome-wide CRISPR screen was performed to identify radioresistance genes in the MDA-MB-231 TNBC cell line. An in vitro clonogenic assay was used to assess antipro-liferative effects of Artemis knockout or pharmacologic inhibition of Artemis, either alone or in combination with RT. Tumor doubling time and animal survival were assessed using an in vivo xenograft model. RNA-Seq analysis was performed to identify genes and pathways deregulated under Artemis knockout conditions, both alone and in combina-tion with RT. Cellular senescence was evaluated using a -galactosidase assay. Our CRISPR screen identified Artemis as a top hit in RT-treated MDA-MB-231 cells, whose depletion led to radiosensitization in TNBC. Artemis knockout significantly reduced cell proliferation and enhanced the antiproliferative effects of RT in vitro. Compared to mice bearing control MDA-MB-231 xenografts, Artemis knockout exhibited prolonged survival that was further enhanced with RT. Bulk RNA-Sequencing indicated that the antipro-liferative and radiosensitization effects of Artemis depletion were mediated by activation of cellular senescence which was confirmed with a -galactosidase assay. Taken together, our results highlight the critical role of Artemis in TNBC cell proliferation and response to radiation. Our findings identify Artemis as a potential biomarker in-dicative of sensitivity to radiation and a putative target that could be inhibited to enhance the efficacy of RT in TNBC.

Homeostasis in the kidney proximal tubule (PT) requires coordination between metabolism and differentiation, yet the mechanisms governing this balance remain elusive. Here, we integrate model organisms, multiomics profiling, and human genetics to identify the autophagy regulator ATG7 as a key determinant of cell-fate decisions, sustaining PT specialization in health and contributing to dysfunction in disease. In mice, PT-specific deletion of ATG7 reprograms differentiated cells into anabolic, proliferative states, impairing their specialized function and causing kidney tubulopathy. Mechanistically, loss of ATG7-dependent autophagy hinders lipid droplet clearance and restricts fatty-acid oxidation (FAO), leading to energy depletion and functional decline. In zebrafish pronephros, re-expression of wild-type ATG7 restores homeostasis in atg7 mutants, while pharmacological FAO inhibition triggers dysfunction. In humans, ATG7 variants associate with cardio-renal-metabolic traits and increased disease risk, whereas low ATG7 expression correlates with transcriptional signatures of metabolic reprogramming, loss of epithelial markers, and poor prognosis in renal cell carcinoma. These findings establish a conserved genetic paradigm that links autophagy to kidney epithelial cell-fate specialization, with implications for disease, cancer, and metabolic health.

Inland recirculating aquaculture is a thriving food industry providing sustainable and locally sourced high-quality protein. However, its expansion is accompanied by emerging challenges regarding the spread of pathogens and diseases. Detection and management of pathogens in aquaculture remain underdeveloped compared to other animal farming sectors due to the vast diversity of species involved, the non-domesticated species, and limited knowledge regarding pathogens, host responses, and disease mechanisms. Furthermore, recirculating aquaculture systems are heavily dependent on beneficial bacterial communities for waste product removal and water quality maintenance, with opportunistic pathogens constituting an inherent component of these microbial communities. To enhance the potential of inland aquaculture as a sustainable source of protein, it is imperative to adopt advanced tools for pathogen detection and monitoring and for assessing the overall health of the microbial ecosystem. This paper presents an overview of promising current molecular and technological advancements that offer solutions for pathogen detection and system monitoring in aquaculture. We focus on recently developed point-of-care and on-site detection methods using miniaturized laboratory equipment and robust workflows that operate independently of cold chain logistics. We explore current methodologies for monitoring pathogens in the environment rather than through fish health assessments. Lastly, we discuss techniques from other scientific disciplines in aquaculture, including CRISPR-Cas protocols for pathogen detection and the implementation of "omics" approaches for comprehensive characterization of microbial states. These methods demonstrate considerable potential for pathogen surveillance and, subsequently, early responses in the dynamic aquaculture field. Through a better understanding of available options aquaculture managers and molecular scientist can collaborate and optimize systems. This paper aims to facilitate communication between molecular scientists and aquaculture managers, equipping the aquaculture industry with knowledge to enhance pathogen management techniques in their facilities.

CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated 9) has revolutionized gene editing technology by offering high precision and efficiency. Despite its powerful potential, significant challenges remain to completely avoid side effects and to improve delivery systems. This review focuses on CRISPR/Cas9 system delivery cargoes and on the different types of delivery vehicles. Emphasis is placed on advantages, disadvantages and gene editing efficiency of each method as well as introducing delivery vehicles such as lipid nanoparticles, which offer potential to enhance precision and reduce immune responses. Additionally, Cas9 aggregation behavior, a factor rarely described in literature, is reviewed due to its potential impact on editing efficiency, an often-overlooked factor that could influence future therapeutic applications. By addressing these challenges, gene editing moves closer to maximizing its therapeutical applications.

Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life [1]. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells, and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV infected and bystander activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing. Importance Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.

ABSTRACT CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture and human therapeutics. However, their dependence on a 5’-TTTV-3’ protospacer-adjacent motif (PAM) next to DNA target sequences restricts Cas12a’s gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a (LbCas12a) with expanded PAM recognition. The resulting Cas12a variants use a range of non-canonical PAMs while retaining recognition of the canonical 5’-TTTV-3’ PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5’-NYHV-3’ PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

DNA plasmids (pDNA) are essential for gene cloning and protein expression, whereby engineered plasmids serve as vectors to insert foreign DNA into host cells, enabling mass production of proteins and vaccines. Furthermore, pDNA is used in CRISPR-based gene editing, RNA therapeutics, and DNA vaccines. Due to the rapidly increasing use and application of a wide variety of pDNA, analytical methods to characterize their key attributes are vital. Because of their high molecular weight, accurate and fast mass analyses of pDNA, as a measure of quality control, is rather challenging. Here we explore mass photometry (MP) to analyze pDNAs and find that it completely fails using standard procedures as developed for MP on proteins, with masses underestimated by 30-40%. Even though the landing of pDNA during MP analysis can be improved by using coated glass slides, the large dsDNA particles diffract light beyond the diffraction limit, rendering most landing events unusable. To overcome these issues, we introduce a fast (30 s) and simple protocol to convert dsDNA particles rapidly into ssDNA-like particles just prior to analysis and show that these particles behave nearly perfect for MP. Using this protocol accurate and correct masses of pDNAs can be obtained by MP, with values within 1-3% of the expected mass. Using this protocol, MP can be used to mass analyze pDNA constructs from 1 to 15 MDa, suggesting that this approach may be widely adopted within academia and biopharma for essentially all plasmids.

Intronic hexanucleotide repeat expansions in the C9orf72 gene represent the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion decreases C9orf72 expression in affected patients, indicating that loss of C9orf72 function (LOF) acts as a pathogenic mechanism. Several models using Danio rerio (zebrafish) for C9orf72 depletion have been developed to explore disease mechanisms and the consequences of C9orf72 LOF. However, inconsistencies exist in reported phenotypes, and many have yet to be validated in stable germline ablation models. To address this, we created a zebrafish C9orf72 knockout model using CRISPR/Cas9. The C9orf72 LOF model demonstrates, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits. Additionally, homozygous C9orf72 LOF larvae exhibited mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we performed a screen on an established Caenorhabditis elegans ( C. elegans ) C9orf72 homologue ( alfa-1 ) LOF model, identifying 12 compounds that enhanced motility, reduced neurodegeneration, and alleviated paralysis phenotypes. Motivated by the shared motor phenotype, 2 of those compounds were tested in our zebrafish C9orf72 LOF model. Pizotifen malate was found to significantly improve motor deficits in C9orf72 LOF zebrafish larvae. We introduce a novel zebrafish C9orf72 knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool for in vivo C9orf72 research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation. Author Summary Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of motor neurons, with no curative treatments currently available. The most common genetic cause is a hexanucleotide repeat expansion in the C9orf72 gene, which reduces its expression and implicates loss-of-function (LOF) as a disease mechanism. However, the complete functions of C9orf72 and its role in ALS remain unclear. Zebrafish models with indirect partial reduction of C9orf72 expression have shown promise in recapitulating key aspects of ALS, but inconsistencies have been observed across these models. To address these challenges, we developed a stable genetic C9orf72 LOF zebrafish model to study the effects of its LOF, validate previous findings, and test potential ALS therapeutics. Our model displays swimming activity deficits, reduced growth, increased mortality, and mild spinal motor neuron abnormalities. We demonstrated that pizotifen malate significantly improved motor function in both our model and a similar well-established worm model. These results underscore the differences between indirect depletion and direct genetic LOF models while identifying pizotifen malate as a promising candidate for preclinical testing. This zebrafish model serves as a valuable tool for understanding C9orf72 -associated ALS mechanisms and advancing therapeutic development.

Abstract RNA displays significant heterogeneity in its structures, dynamics, and functional roles within cells. CRISPR-based imaging methods provide a powerful approach to visualize RNA molecules in living cells. However, the expression of constitutive fluorescence modules in traditional CRISPR-based tools always produce high background and nonspecific signals. To address these challenges, we developed a switched fluorescent CRISPR-tDeg (CtDeg) system that minimizes background for RNA imaging in living cells. This CtDeg system consists of a signal module-deactivated Cas13 protein (dCas13)-degron complex and an engineered switched sgRNA. The signal module-fused complex is degraded unless it binds to the engineered sgRNA, which is activated upon targeting the desired RNA. This interaction will generate a stable signal module-dCas13: sgRNA complex, enabling specific imaging of RNA in living cells. Using the CtDeg system, we visualized the movement and assembly dynamics of paraspeckles at the RNA level. Additionally, we tracked SARS-CoV-2 genome dynamics at the early stage after viral invasion for the first time, providing direct imaging data that demonstrate SARS-CoV-2 infection up-regulates the expression levels of the disease-associated NEAT1 gene, thereby filling a gap in this research area. The CtDeg system represents a powerful tool for specific imaging of RNA to reveal the underlying functions and interactions within living cells.

Abstract Background Vibrio sp. dhg is a fast-growing, alginate-utilizing, marine bacterium being developed as a platform host for macroalgae biorefinery. To maximize its potential in the production of various value-added products, there is a need to expand genetic engineering tools for versatile editing. Results The CRISPR-based cytosine base editing (CBE) system was established in Vibrio sp. dhg, enabling C:G-to-T:A point mutations in multiple genomic loci. This CBE system displayed high editing efficiencies for single and multiple targets, reaching up to 100%. The CBE system efficiently introduced premature stop codons, inactivating seven genes encoding putative restriction enzymes of the restriction-modification system in two rounds. A resulting engineered strain displayed significantly enhanced transformation efficiency by up to 55.5-fold. Conclusions Developing a highly efficient CBE system and improving transformation efficiency enable versatile genetic manipulation of Vibrio sp. dhg for diverse engineering in brown macroalgae bioconversion.

Abstract The CRISPR/Cas12a system is known for its intrinsic RNA-guided trans -cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of "Pseudo Hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single crRNA and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the PAM-distal region. It can also detect ultrashort RNA sequences as short as 6–8 nucleotides and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and HPV16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.

Abstract Warming trend of the climate has been linked with yield losses in many crops. Like other cereals, rice is highly sensitive to above-typical temperatures during reproductive and grain filling stages. In fact, nighttime temperatures have risen faster than daytime temperatures in many parts of the world. For this reason, rice studies in recent years have focused on the effect of high nighttime temperature (HNT) on grain yield. Several studies have shown that HNT disturbs key processes in reproductive development and grain filling that lead to reduced spikelet fertility (SF) and enhanced grain chalkiness (Srivastava et al., 2024). Chalkiness is the opaque area on the grain, and it is not just an appearance issue, it also impacts milling quality. Above the generally acceptable chalk values (6-10%), every 1% increase in chalkiness leads to 1% decline in head rice yield (HRY) (Zhao and Fitzgerald 2013). Therefore, breeding HNT tolerance is vital for safeguarding grain yields from heat waves in the future. However, breeding efforts have been impeded by the lack of reliable tolerance alleles in modern cultivars. Breeding is also complicated by the complex nature of HNT tolerance as not only SF and grain quality traits, but other yield components such as panicle length, grain width, grain size, and grain weight are also affected by HNT in a genotype-dependent manner. Not surprising, hundreds of QTLs have been identified in the genomics studies. Of which, Chalk5 (Os05g0156900) is most notable as it stands as one of the few functionally validated QTL (Fan et al., 2024; Gann et al., 2023; Li et al., 2014).

ABSTRACT As an abundant fungal colonizer of human skin, Malassezia has long been associated with pathological skin conditions, yet its role in skin homeostasis remain poorly understood. Here, we demonstrate that Malassezia furfur plays an active role in maintaining epidermal integrity by producing tryptophan-derived metabolites that activate the aryl hydrocarbon receptor (AhR), a key regulator of keratinocyte differentiation and inflammation. Using a fungal mutant defective in indole production, we show that M. furfur -derived AhR activation is required to restore barrier function and control inflammation in diseased skin. AhR-deficient mice fail to benefit from M. furfur -mediated barrier protection, underscoring the importance of microbial-derived AhR agonists in skin physiology. These findings establish a previously unrecognized mutualistic role for Malassezia in epidermal homeostasis, challenging its perception as solely a pathogenic fungus and expanding our understanding of the skin microbiota’s influence on barrier function and immune regulation. KEY FINDINGS Malassezia -derived indoles reprogram epidermal gene expression to enhance keratinocyte function. AhR activation by Malassezia restores skin barrier integrity and reduces inflammation. Malassezia Sul1-dependent tryptophan metabolism is essential for the production of AhR agonists. The barrier protective effects of Malassezia are mediated specifically through keratinocyte intrinsic AhR signaling.

Gastrointestinal (GI) dysfunction emerges years before motor symptoms in Parkinson’s disease (PD), implicating the enteric nervous system (ENS) in early disease progression. However, the mechanisms linking the PD hallmark protein, α-synuclein (α-syn), to ENS dysfunction - and whether these mechanisms are influenced by inflammation - remains elusive. Using iPSC-derived enteric neural lineages from patients with α-syn triplications, we reveal that TNF-α increases mitochondrial-α-syn interactions, disrupts the malate-aspartate shuttle, and forces a metabolic shift toward glutamine oxidation. These alterations drive mitochondrial dysfunction, characterizing metabolic impairment under cytokine stress. Interestingly, targeting glutamate metabolism with Chicago Sky Blue 6B restores mitochondrial function, reversing TNF-α-driven metabolic disruption. Our findings position the ENS as a central player in PD pathogenesis, establishing a direct link between cytokines, α-syn accumulation, metabolic stress and mitochondrial dysfunction. By uncovering a previously unrecognized metabolic vulnerability in the ENS, we highlight its potential as a therapeutic target for early PD intervention.

BREX ( B acte r iophage Ex clusion) systems, identified through shared identity with Pgl ( P hage G rowth L imitation) systems, are a widespread, highly diverse group of phage defence systems found throughout bacteria and archaea. The varied BREX Types harbour multiple protein subunits (between four and eight) and all encode a conserved putative phosphatase (PglZ aka BrxZ) and an equally conserved, putative ATPase (BrxC). Almost all BREX systems also contain a site-specific methyltransferase (PglX aka BrxX). Despite having determined the structure and fundamental biophysical and biochemical behaviours for the PglX methyltransferase, the BrxL effector, the BrxA DNA-binding protein and the BrxR transcriptional regulator, the mechanism by which BREX impedes phage replication remains largely undetermined. In this study, we identify a stable BREX sub-complex of PglZ:BrxB, validate the structure and dynamic behaviour of that sub-complex, and assess the biochemical activity of PglZ, revealing it to be a metal-dependent nuclease. PglZ can cleave cyclic oligonucleotides, linear oligonucleotides, plasmid DNA and both non-modified and modified linear phage genomes. PglZ nuclease activity has no obvious role in BREX-dependent methylation, but does contribute to BREX phage defence. BrxB binding does not impact PglZ nuclease activity. These data contribute to our growing understanding of the BREX phage defence mechanism.

CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis , after which it remains catalytically active and non-specifically cleaves ssDNA in trans . Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli , and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied ‘NUC loop’, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract

Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Most research focusses on Dictyostelium discoideum, which - due to its ease of culturing and genetic tractability - has been adopted as a model species in the fields of developmental biology, cell biology and microbiology. Over decades, genome editing methods in D. discoideum have steadily improved but remain relatively time-consuming and limited in scope, effective in a few species only. Here, we introduce a CRISPR-Cas9 editing protocol that is cloning-free, selection-free, highly-efficient, and effective across Dictyostelid species. After optimizing our protocol in D. discoideum, we obtained knock-out efficiencies of ~80% and knock-in efficiencies of ~30% without antibiotic selection. Efficiencies depend on template concentrations, insertion sizes, homology arms and target sites. Since our protocol is selection-free, we can isolate mutants as soon as one day post-transfection, vastly expediting the generation of knock-outs, fusion proteins and expression reporters. Our protocol also makes it possible to generate several knock-in mutations simultaneously in the same cells. Boosted by cell-sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing protocol to phylogenetically distant Dictyostelid species, which diverged hundreds of millions of years ago and have never been genome edited before. Our protocol therefore opens the door to performing broad-scale genetic interrogations across Dicyostelids.

We introduced two targeted DNA double-strand breaks on the same Arabidopsis chromosome using CRISPR-Cas9 and replaced the FLOWERING LOCUS T ( FT ) promoter with that of a histone variant gene through chromosomal inversion. The resulting lines misexpressed FT and flowered early, like FT -overexpressing transgenic plants. This system can be used to create gain-of-function mutations that modify target gene expression as desired without incorporating foreign DNA sequences.

ABSTRACT Insufficient infiltration of cytotoxic lymphocytes to solid tumors limits the efficacy of immunotherapies and cell therapies. Here, we report a programmable mechanism to mobilize Natural Killer (NK) and T cells to breast cancer tumors by engineering these cells to express orphan and metabolite-sensing G protein-coupled receptors (GPCRs). First, in vivo and in vitro CRISPR activation screens in NK-92 cells identified GPR183 , GPR84 , GPR34 , GPR18 , FPR3 , and LPAR2 as top enhancers of both tumor infiltration and chemotaxis to breast cancer. These genes equip NK and T cells with the ability to sense and migrate to chemoattracting metabolites such as 7α,25-dihydroxycholesterol and other factors released from breast cancer. Based on Perturb-seq and functional investigations, GPR183 also enhances effector functions, such that engineering NK and CAR NK cells to express GPR183 enhances their ability to migrate to, infiltrate, and control breast cancer tumors. Our study uncovered metabolite-based tumor immune recruitment mechanisms, opening avenues for spatially targeted cell therapies.

Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation. Significance Obesity and its associated metabolic disorders pose significant public health challenges, yet the molecular mechanisms regulating adipocyte function remain incompletely understood. Small open reading frames (smORFs) and their encoded microproteins represent an emerging class of regulatory elements with potential roles in metabolism. Here, we leveraged CRISPR/Cas9 screening to functionally characterize smORFs in adipocytes, identifying novel regulators of cell proliferation and lipid metabolism. Our findings demonstrate that conservation is not a prerequisite for smORF function, as we validated a mouse-specific microprotein that modulates adipocyte differentiation. This work establishes a robust pipeline for unbiased smORF discovery and highlights the potential for species-specific microproteins to regulate adipose biology. Future studies in human adipocytes may uncover additional microproteins with therapeutic relevance for obesity and metabolic disease.

Summary The ability to maintain invariant developmental phenotypes across disparate environments is termed canalization, but few examples of canalization mechanisms are described. In plants, robust flower production across environmental gradients contributes to reproductive success and agricultural yields. Flowers are produced by the shoot apical meristem (SAM) in an auxin-dependent manner following the switch from vegetative growth to the reproductive phase. While the timing of this phase change, called the floral transition, is sensitized to numerous environmental and endogenous signals, flower formation itself is remarkably invariant across environmental conditions. Previously we found that CLAVATA peptide signaling promotes auxin-dependent flower primordia formation in cool environments, but that high temperatures can restore primordia formation through unknown mechanisms. Here, we show that heat promotes floral primordia patterning and formation in SAMs not by increased auxin production, but through the production of the mobile flowering signal, florigen, in leaves. Florigen, which includes FLOWERING LOCUS T ( FT ) and its paralog TWIN SISTER OF FT ( TSF ) in Arabidopsis thaliana , is necessary and sufficient to buffer flower production against the loss of CLAVATA signaling and promotes heat-mediated primordia formation through specific SAM expressed transcriptional regulators. We find that sustained florigen production is necessary for continuous flower primordia production at warmer temperatures, contrasting florigen’s switch-like control of floral transition. Lastly, we show that CLAVATA signaling and florigen synergize to canalize flower production across broad temperature ranges. This work sheds light on the mechanisms governing the canalization of plant development and provides potential targets for engineering crop plants with improved thermal tolerances.

Abstract Glioblastoma remains an incurable and highly aggressive brain tumor due to its immune-suppressive microenvironment. In this paper, we propose a novel theoretical approach using CRISPR-engineered macrophages to infiltrate, recognize, and eradicate glioblastoma.