Precision genome editing, particularly using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is advancing crop improvement by enabling targeted and efficient genetic modifications. Root and tuber crops such as potato, cassava, sweet potato, and yam are vital for global food and nutritional security but remain highly vulnerable to climate change, pests, diseases, and limited genetic diversity. Genome editing technologies facilitate the development of improved traits, including enhanced disease resistance, tolerance to abiotic stress, improved nutritional quality, and extended shelf life. This review synthesizes recent advances in genome editing for root and tuber crops across global production systems, including illustrative examples from Sub-Saharan Africa, where active genome editing initiatives are being implemented. It further examines key technical constraints, such as low efficiency of plant transformation and regeneration, and highlights regulatory challenges arising from differing policy frameworks across countries. Emerging solutions are discussed, including genotype-independent editing strategies and DNA-free approaches that avoid the integration of foreign genetic material. Addressing these challenges will be critical for developing resilient and sustainable food systems. Unlike previous reviews, this study integrates mechanistic insights with cross-crop synthesis and proposes next-generation genome editing strategies for engineering complex traits in polyploid root and tuber crops.

NAA25 as a regulator of insulin signaling: Integration of FOXO1 imaging CRISPRi screen and Mendelian randomization.

Cyanobacterial carbonic anhydrases drive carbon recycling and prodrug biosynthesis in Escherichia coli.

Atrial fibrillation (AF), the most common sustained arrhythmia, has a complex genetic basis; however, the molecular mechanisms linking rare and common variants remain poorly understood. Polygenic risk score (PRS) analysis in the UK Biobank and All of Us cohorts reveals that carriers of protein-altering LMNA variants (PAVs) have a significantly higher risk of incident AF than predicted by PRS alone, supporting an additive effect of common polymorphisms and LMNA variants. Induced pluripotent stem cell derived atrial cardiomyocytes (iPSC-aCMs) from individuals carrying the pathogenic missense variant p.S143P in LMNA exhibit widespread disruption of chromatin architecture and perturbation of atrial gene regulatory networks, particularly at loci harboring AF-associated variants and transcription factors essential for atrial rhythm control and contractility. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenetic editing validates the function of several AF-associated regulatory elements and their downstream targets. Notably, reduced accessibility at an intronic SCN10A enhancer harboring the AF-associated SNP rs6801957 is associated with reduced sodium current in p.S143P iPSC-aCMs. These findings are reproduced in iPSC-aCMs derived from an additional individual carrying a distinct pathogenic LMNA variant, supporting a broader mechanism in which rare LMNA variants and common polymorphisms converge on shared regulatory networks to influence AF susceptibility and highlighting the value of integrating both in arrhythmia risk assessment.

CRISPR-Cas systems have emerged as a versatile tool for diagnosing, treating, and preventing infectious diseases. This review highlights translational advancements in CRISPR-Cas-based applications, concentrating on the past decades in diagnostics, therapeutic genome editing, and vaccine development. The article highlights key platforms like DETECTR and SHERLOCK, which enable rapid, sensitive pathogen detection, and explores CRISPR-Cas9 systems in therapeutic strategies for directly targeting viral genomes and combating antimicrobial resistance. It also examines the role of CRISPR-Cas9 in engineering live-attenuated and personalized neoantigen vaccines. Principal findings demonstrate a clear progression from experimental proof-of-concept to preclinical applications primarily in CRISPR-based diagnostics and the engineering of live-attenuated vaccine candidates, whereas translation in CRISPR-based therapeutics and personalized neoantigen vaccines for infectious diseases remains at earlier, more exploratory stages. CRISPR-based diagnostics have progressed further toward clinical evaluation than therapeutics due to delivery and safety constraints, while personalized neoantigen vaccines are included mainly as an emerging, comparative concept for infectious diseases rather than a mature application. This review uniquely integrates CRISPR-based diagnostics, therapeutics, and vaccine development within a single infectious disease framework, critically assesses their current maturity, and systematically highlights technical, regulatory, and ethical barriers alongside realistic future priorities. The review concludes that while CRISPR-Cas holds transformative potential for infectious disease management, significant challenges in delivery efficiency, off-target effects, and ethical regulation must be addressed to ensure safe and equitable clinical translation.

CRISPR-Cas systems are prokaryotic immune mechanisms often targeted by phage-encoded anti-CRISPR (Acr) proteins. This study characterizes AcrIF19, a potent inhibitor of the type I-F system in Pectobacterium atrosepticum. The cryo-EM structure of the apo Cas2/3 and Cas2/3-AcrIF19 complex reveals a dual inhibitory mechanism. AcrIF19 employs a negatively charged β2-β3 loop to sterically occlude the non-target DNA strand entry channel, acting as a competitive inhibitor to disrupt Cas2/3 recruitment. Concurrently, this steric occlusion impedes ssDNA-mediated allosteric activation, which locks the critical helix-like loop motif in an inhibitory conformation and thereby abrogates DNA cleavage activity. AcrIF19 represents an anti-CRISPR protein inhibiting Cas2/3 via two different mechanisms, integrating a competitive ssDNA inhibitor with an allosteric blockade to suppress both target recruitment and DNA cleavage.

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater detection results obtained using HICAS-Chip showed high concordance (95.83%) with RT-qPCR assays. This platform provides an efficient portable device for the early detection and continuous monitoring of HFMD epidemic trends by wastewater-based epidemiology.

Transitioning to a carbon-neutral economy requires biocatalysts that can efficiently convert waste-derived substrates into valuable products. Acetogens are industrially relevant organisms for gas fermentation, but the lack of genetic toolkits tailored to their physiology has constrained metabolic engineering. We present the first synthetic promoter-CRISPRi platform specifically optimized for Eubacterium callanderi KIST612, a model acetogen with high industrial potential. This system provides tunable and predictable regulation of gene expression, extending from mild repression to a near-complete knockdown that could alternate gene deletion systems. This system could be used for not only advancing fundamental understanding of acetogen physiology but also providing a broadly applicable genetic toolbox for precision engineering of sustainable microbial biorefineries.

Cellular-state control using ribozyme-scaffolded miRNA-sensing and CRISPR-mediated actuation.

Limosilactobacillus fermentum is a versatile lactic acid bacterium with significant probiotic and biotechnological potential, yet the genomic determinants underlying its ecological adaptation and therapeutic applications remain underexplored. This study performed a comparative genomic analysis of 52 L. fermentum strains, with a focus on the novel strain ATT-06 isolated from traditional Turkish shalgam. We uncovered substantial genetic diversity, evidenced by an open pan-genome (18,647 genes) and a small core genome (718 genes). CRISPR-Cas systems were prevalent (46 strains), with Type-IE and Type-IIA being most common and mutually exclusive from Type-IC. Strain ATT-06, which uniquely produced gamma-aminobutyric acid at 17.74µg/mL, harbored a Type-IIA CRISPR system and a single prophage. In silico molecular docking and dynamics simulations revealed that the bacteriocin Lactococcin, encoded by strain ATA-LTC-Lf170503, exhibited strong binding affinities (ΔG: -8.1 to -13.0kcal/mol) against Rho proteins of Staphylococcus aureus and Escherichia coli, outperforming Acidocin A. These findings highlight the genomic plasticity and adaptive mechanisms of L. fermentum, and position strain ATT-06 as a promising probiotic candidate with potential neuroactive and antimicrobial applications.

CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to nonmodel strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three nonmodel strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across nonmodel E. coli strains, providing guidelines for diverse applications of these tools.

Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel–Lindau–deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α–dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy–driven enhancer activation, this work defines a trans-acting cytokine–JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.

Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.

Exponential signal amplification through coupled rolling circle transcription and CRISPR/Cas13a cleavage for ultrasensitive molecular diagnostics.

Antimicrobial resistance (AMR) poses a significant threat to global health by diminishing the effectiveness of conventional antibiotics. This study aims to assess, using a systems biology approach, a potential synthetic biology-based strategy that employs engineered conjugative probiotic bacteria and bacteriophages to combat AMR, examining the implications of implementing targeted gene silencing as an alternative to direct bacterial killing. A comprehensive mathematical model was developed to describe the dynamics of the delivery systems (engineered conjugative probiotic bacteria and engineered phages) and the antimicrobial actuators being studied (genome cutting via CRISPR systems and AMR-gene silencing through CRISPR interference), also compared to traditional phage therapy (selection of phages capable of killing pathogens through bacterial-specific viral infection). The target population includes antibiotic-resistant bacteria competing with other probiotic bacteria for colonizing the host environment. The model explicitly incorporates parameters for mutations that affect actuator functionality and simulates their impact on overall therapeutic performance. Simulations show how variations in actuator efficiency and emergence of new mutations in target pathogens affect the long-term suppression of resistance genes. Including mutational effects provides insights into system robustness and guides optimal therapeutic design choices. The proposed modeling framework effectively captures key biological and mechanistic aspects of engineered therapies, enabling the prediction and optimization of each intervention against resistant pathogens. It highlights engineered phages and CRISPR interference as the most promising candidates for the design of new engineered biological therapeutics. This study establishes a quantitative foundation for rational design and dosage optimization in engineered phage- and bacterial-based therapies, advancing the use of synthetic biology methods to fight antimicrobial resistance.

The transition from indeterminate to determinate growth represents a key achievement in crop improvement, as it enhances agricultural productivity by synchronizing flowering, facilitating uniform harvest, and improving overall efficiency. In tomato and other crops, this shift is largely governed by mutations in the SELF-PRUNING (SP) gene, a key member of the CENTRORADIALIS (CEN), TERMINAL FLOWER 1 (TFL1), and SELF-PRUNING (SP) (CETS) gene family that regulates the vegetative to reproductive phase transition and influences overall shoot architecture. With increasing labour constraints, climate variability and rising global food security challenges, the ability to engineer optimized plant architectures has become increasingly important. CRISPR-based genome editing provides a precise and efficient strategy to modify SP/TFL1 homologs, enabling targeted transition from indeterminate to compact, determinate growth forms that exhibit synchronized flowering and enhanced mechanical harvestability. These genome editing approaches have been successfully applied across diverse crop species, including tomato, legumes, cotton, cereals and horticultural crops. This review consolidates current understanding of the molecular mechanisms governing determinacy, with emphasis on the central role of SP/TFL1 genes and their interactions with hormonal pathways such as auxin and cytokinin. By integrating these insights with recent advances in CRISPR-based editing platforms, this review provides a practical framework for researchers and breeders aiming to leverage CRISPR technology for next-generation crop improvement. Such strategies hold significant potential for enhancing productivity, resilience and sustainability within modern agricultural systems.

This study identified family with sequence similarity 205, member A (FAM205A) as a clinically significant acrosomal protein critically implicated in male idiopathic infertility. Integrated proteomic analysis of sperm from in vitro fertilization (IVF) patients categorized by fertilization outcome (n = 12 per group; clinical validation, n = 267) revealed a strong correlation between FAM205A expression and fertilization success (76.3% in high-expression groups vs 15.4% in low-expression groups, P < 0.05), independent of standard semen parameters. Functional analyses utilizing pooled human sperm samples (n = 20 per group) revealed that low FAM205A expression led to a significantly decreased calcium-induced acrosome reaction response (28.9% vs 49.2% in high FAM205A expression, P < 0.01), a phenotype corroborated by anti-FAM205A antibody blockade (38.1% inhibition, P < 0.01). The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) genome editing system (CRISPR-Cas9)-generated knockout mice lacking Fam205a1, which is the murine ortholog of human FAM205A, exhibited complete infertility, characterized by oligospermia, asthenozoospermia, teratozoospermia, disrupted acrosome formation, and significantly reduced sperm motility. Mechanistic studies in Fam205a1 knockout mice revealed that FAM205A deficiency induces spermatid apoptosis and impairs acrosomal exocytosis, a crucial process for sperm-egg fusion. The results demonstrate that FAM205A regulates fertility through two mechanisms: preserving structural integrity during spermiogenesis and facilitating the acrosomal reaction (AR). This study identified FAM205A as a reliable diagnostic biomarker for idiopathic male infertility and a potential treatment target in reproductive medicine.

The rising rate of drug-resistant fungal infections and the emergence of intrinsically resistant pathogens pose growing clinical challenges. Because fungi are closely related to mammals, developing antifungals without toxic off-target effects is difficult. Targeted gene repression can model drug-mediated inhibition and reveal gene dosage sensitivity, but traditional approaches in the fungal pathogen Candida albicans are labour intensive and low throughput. Here we adapt pooled CRISPR interference (CRISPRi) screening in C. albicans to enable large-scale functional genomic analysis. We assess repression sensitivity of 130 essential genes conserved in fungi without close homologues in humans and identify highly dosage-sensitive genes across multiple pathways. Screening across ten environmental conditions reveals environment-dependent effects on gene sensitivity. Extending these experiments to two drug-resistant clinical isolates shows that many fitness defects are conserved across genetic backgrounds. Thus, CRISPRi pooled screening enables rapid, large-scale functional genomics across diverse genetic backgrounds in C. albicans.

The genus Priestia has recently gained attention for its plant growth-promoting potential. To examine the genomic traits and biosafety profile for potential field application as a native, climate-smart bioinoculant, we sequenced, assembled and annotated the genome of Priestia megaterium strain MHES4, isolated from the rhizosphere of tomato plant grown in drought-prone ecosystem of Rajshahi, Bangladesh. Genome assembly data from the shotgun whole genome sequencing (WGS) of the P. megaterium MHES4 revealed 60 contigs with a total length of 5,267,048bp, an N50 of 446,003bp and 37.9% G + C content. The mean sequencing depth was 127.58×, with 100% breadth of coverage. Genome completeness assessed was 97.43% with 3.5% contamination, confirming high assembly quality. In total, 5,484 protein-coding genes were annotated. Additionally, 5,445 protein-coding sequences, 28 tRNAs, and 5 rRNAs were identified. Functional analysis identified gene clusters involved in the synthesis of secondary metabolites, such as phytoene synthase and alpha-amylase, and a Type I CRISPR-Cas system. Biosafety assessment using in silico tools detected no virulence factors or transmissible antibiotic resistance genes, indicating its potential safe use in agriculture. Overall, this genomic resource provides valuable insights into the genetic potential of P. megaterium MHES4 for nutrient cycling and adaptation to the rhizosphere environment.

BackgroundColorectal cancer (CRC) is the third most common malignancy worldwide, and the role of E2F transcription factor 6 (E2F6) in CRC remains controversial.MethodsWe analyzed E2F6 mRNA expression across 19 platforms (2,449 CRC patients and 1,328 controls), evaluated expression patterns using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, assessed E2F6 dependency using clustered regularly interspaced short palindromic repeats (CRISPR) knockout data from 52 CRC cell lines, and validated protein expression by immunohistochemistry (IHC) in 200 paired CRC and adjacent tissues. Associations between E2F6 and clinicopathological features were analyzed.ResultsE2F6 was significantly upregulated in CRC versus controls (summary receiver operating characteristic (sROC) area under the curve (AUC) = 0.93), supported by scRNA-seq and spatial transcriptomics. E2F6 knockout suppressed proliferation across CRC cell lines, and IHC confirmed higher E2F6 protein expression (AUC = 0.91). Elevated E2F6 correlated with adverse clinicopathological features including female sex, age ≥ 60 years, advanced T stage, high-grade tumor budding, and higher histological grade.ConclusionsE2F6 is highly expressed in CRC and is associated with unfavorable clinicopathological features, supporting its potential utility as a diagnostic biomarker and a candidate target for CRC stratification and therapy development.