Search Results (67)

Endogenous gene tagging using CRISPR has changed the understanding of the role played by different proteins due to the ability to track and study proteins in their natural state. With CRISPR-based gene tagging, it is possible to insert fluorescent, luminescent, epitope, affinity, and proximity labels into the target protein at its endogenous genomic location without affecting its physiological expression and dynamics. Here, we discuss the DNA-repair mechanisms employed in endogenous gene tagging, including homology-dependent repair, NHEJ-based integration, and alternative approaches that can be used with challenging cell types. Key aspects of efficient CRISPR tagging experiments are also described. Additionally, we review recent advances in the increasing array of protein tag technologies, including fluorescent proteins, split-reporter technologies, NanoLuc/HiBiT, peptide epitopes, and proximity biotinylation enzymes. Lastly, we review the scalability of endogenous tagging approaches using multiplex editing, atlas-scale proteome tagging, iPSC-based disease modeling, and drug discovery platforms for assessing target engagement, protein degradation, phenotype screening, and mechanism of action of compounds. Although difficult in primary and pluripotent cells, new methods based on avoiding double-strand breaks, such as prime editing, PASTE, and CRISPR associated transposases, will drive the future expansion of endogenous tagging approaches. Such developments firmly set up CRISPR gene tagging as a fundamental technology in quantitative cell biology and translational pharmacology. Full article

Background/Objectives: Immune checkpoints are critical regulatory pathways that maintain peripheral tolerance and prevent autoimmunity. Among these, the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) axis serves as a major inhibitory pathway that terminates T cell responses. While protein-based checkpoint blockade (ICB) targeting this axis has revolutionized clinical cancer therapy, its clinical efficacy is frequently limited by low response rates, immune-related adverse events (irAEs), and the emergence of adaptive resistance. To break through these bottlenecks, genetic interruption has emerged as a high-precision alternative to modulate the PD-1/PD-L1 pathway at the nucleotide level. Methods: A comprehensive systematic review of literature was performed across major databases (PubMed, Web of Science), with a focus on high quality studies published up to 2026. Results: Direct genomic disruption via CRISPR/Cas9 and post-transcriptional silencing through RNA interference can effectively neutralize inhibitory signaling at its source. Recent advances demonstrate that targeting upstream regulatory nodes—including metabolic checkpoints (e.g., lactate metabolism) and biophysical mechanisms (e.g., liquid–liquid phase separation)—provides superior transcriptional control over PD-L1. Furthermore, engineering CAR-T cells with multiplex gene editing (e.g., TCR/B2M/PD-1 knockout) or localized scFv secretion significantly enhances antitumor potency while reducing systemic toxicity. Innovations in organ-targeted lipid nanoparticles and stimuli-responsive biomimetic carriers further address the delivery barriers in solid tumors. Conclusions: Gene therapy provides a high-precision platform for PD-1/PD-L1 modulation, offering a viable strategy to overcome adaptive resistance. Future clinical application depends on the refinement of safer editing tools, such as base editing, and the standardization of intelligent delivery systems to ensure controllable and scalable cancer immunotherapy. Full article

Background: Upland cotton (Gossypium hirsutum) is a major natural fiber crop and an important model for studying genome evolution and gene function in polyploid plants. However, its large and highly redundant genome presents substantial challenges for efficient and coordinated multiplex genome editing. Methods: Here, we developed a high-efficiency CRISPR/Cas12a-based multiplex genome editing system in cotton by integrating a tRNA–crRNA polycistronic expression strategy with a Bean yellow dwarf virus (BeYDV)-derived replicon. Results: This platform enabled coordinated expression of multiple crRNAs and simultaneous targeting of 16 loci within a centromere-proximal region of chromosome D03 (18.65–24.47 Mb). In individual transgenic lines, up to 10 target sites were edited concurrently, with nine targets exhibiting editing efficiencies above 56% and the highest efficiency reaching 96.46%. High-density multiplex editing predominantly induced small insertions and deletions at target loci. Notably, edited plants exhibited reduced growth and pronounced cytological abnormalities, including chromosome bridges, lagging chromosomes, and abnormal meiotic products. Transcriptome analysis revealed widespread dysregulation of genes involved in chromosome segregation and cell cycle regulation. Despite these functional perturbations, HiFi long-read sequencing detected no large-scale chromosomal rearrangements, indicating that genome instability arises from cumulative local perturbations rather than global structural alterations. Conclusions: Together, our results establish an efficient multiplex genome editing platform in cotton and highlight potential constraints of high-density editing on genome stability in complex plant genomes. Full article

Background/Objectives: Escherichia coli is the predominant pathogen in community-onset urinary tract infections (UTIs) requiring hospitalization. This study characterized antimicrobial resistance profiles, biofilm formation, extended-spectrum β-lactamase (ESBL) gene distribution, and phylogenetic background of E. coli isolates from hospitalized UTI patients in Western Mexico. Methods: Seventy isolates (September 2023–September 2024) underwent susceptibility testing (CLSI M100, 35th edition), multiplex PCR for blaTEM, blaCTX-M, and blaSHV genes, crystal violet biofilm quantification, and Clermont quadruplex PCR phylotyping. Associations were evaluated by Fisher’s exact test with Benjamini–Hochberg FDR (BH-FDR) correction. Results: ESBL phenotype and MDR were detected in 57.1% and 58.6% of isolates. After BH-FDR correction, ESBL production was significantly associated with amikacin (OR = 5.55; 95% CI: 1.80–18.74; q = 0.002) and TMP-SMX non-susceptibility (OR = 3.00; 95% CI: 1.02–9.23; q = 0.036); ciprofloxacin non-susceptibility was linked to MDR status (OR = 7.21; 95% CI: 1.28–75.66; q = 0.017) but not ESBL phenotype. Biofilm was detected in 77.1% of isolates. blaTEM predominated among ESBL producers (85.0%). Phylogroup B2 (51.4%) was inversely associated with recurrent UTI on both univariate (OR = 0.17; 95% CI: 0.03–0.73; p = 0.008) and adjusted analysis (adjusted OR = 0.19; 95% CI: 0.05–0.81; p = 0.025). Phylogroup C (22.9%) exhibited the highest MDR prevalence (81.3%) and the highest biofilm formation rate among phylogroups (87.5%). Conclusions: The high prevalence of ESBL-producing and MDR E. coli, combined with an unexpected predominance of blaTEM, reveals a distinctive local resistance landscape diverging from regional trends. The inverse association of phylogroup B2 with recurrence and TMP-SMX resistance reinforces the clinical value of phylogenetic surveillance in guiding UTI management strategies. Full article

Plant diseases remain a major constraint to crop productivity and global food security. Conventional breeding has long been used to develop resistant cultivars through the introgression of resistance traits from wild relatives and the selection of favorable phenotypes. However, this process is often slow and limited by linkage drag, known genetic diversity, intrinsic genetic limitations, and the rapid evolution of pathogen populations. Molecular breeding strategies, including marker-assisted selection and genomic selection, have improved the precision of resistance breeding but still rely on existing genetic variation. Recent advances in genome editing technologies are transforming plant breeding by enabling precise modification of gene targets. CRISPR-based systems allow targeted gene knockouts, promoter editing, allelic replacement, and multiplex editing to rapidly generate resistance traits. Many studies have demonstrated that editing susceptibility genes or regulatory regions can enhance resistance to diverse pathogens. Recent research shows that resistance can also be improved by targeting non-classical genes involved in plant immunity, including transcription factors, membrane transporters, heat shock proteins, cell wall-related genes, metabolic enzymes, and epigenetic regulators. Emerging tools such as base editing, prime editing, regulatory tools, and transposon-associated genome engineering systems are further expanding the precision and versatility of plant genome editing. Despite these advances, challenges related to delivery systems, editing efficiency, regulatory frameworks, and field validation remain. Continued technological progress and improved knowledge of plant immune networks will be essential to fully integrate genome editing into crop improvement programs. Full article

Tomato brown rugose fruit virus (ToBRFV) poses a major threat to global tomato (Solanum lycopersicum) production, as it can overcome conventional resistance genes that are effective against tobamoviruses. In this study, a multiplex CRISPR/Cas9 system was developed to target the SlTOM1 susceptibility gene family (SlTOM1a–d), which encodes host factors essential for tobamovirus replication. Six guide RNAs (gRNAs), designed following 12 off-target analyses, were assembled into a multiplex CRISPR/Cas9 construct using a Golden Gate cloning strategy and introduced into tomato genotypes through an Agrobacterium-based tissue culture transformation procedure. Although primary T₀ transformants exhibited chimeric mutation patterns, stable inheritance and segregation of edited alleles were confirmed in the T₁ generation. Sequence analyses identified diverse indel mutations across target loci, with SlTOM1d exhibiting the highest editing efficiency. Multiplex genome editing successfully generated single-, double-, and triple-mutant combinations, with higher-order mutants displaying the strongest tolerance phenotypes. Following mechanical ToBRFV inoculation, edited T₁ plants exhibited markedly reduced symptom severity, low viral accumulation, and improved fruit health compared to wild-type controls. RT-qPCR analysis further confirmed significantly reduced viral RNA levels, supporting a host-factor-mediated tolerance mechanism. Importantly, edited lines maintained normal growth and agronomic performance. Collectively, these findings demonstrate that multiplex CRISPR/Cas9-mediated targeting of SlTOM1 homologs represents a promising and practical strategy for improving ToBRFV tolerance in tomato breeding programs. Full article

Despite significant advancements in CRISPR/Cas-based genome editing technology over the past decade, achieving simultaneous homozygous gene editing at multiple targets in primary cells remains a major challenge. In this study, we developed and constructed a CRISPR multi-gene targeting system that integrates episomal vectors with tRNA–sgRNA array technology. This approach leverages scaffold/matrix attachment region (S/MAR) sequences to enable sustained episomal expression of both Cas9 and single-guide RNAs (sgRNAs) without genomic integration, thereby enhancing gene editing efficiency. For simultaneous editing of multiple loci, we used the tRNA–sgRNA architecture to process multiple sgRNAs from a single vector. Using this system in porcine fetal fibroblasts, we achieved concurrent editing of six genes, namely ANXA7, GSK3A, ENTPD6, SIRT3, CYP20A1, and SOCS2, in individual cells. These edited cells supported normal development following somatic cell nuclear transfer, yielding blastocysts with unaltered developmental competence. Collectively, our findings establish a framework for the application of CRISPR/Cas9 in gene-edited pigs, facilitating the generation of multi-gene-edited animals for biomedical and agricultural applications. Full article

Recent advances in gene therapy have highlighted the potential of CRISPR-based gene-editing systems combined with adeno-associated virus (AAV) vectors. However, the limited packaging capacity of AAV remains a significant challenge for the simultaneous expression of Cas effector proteins and guide RNAs within a single vector. To address this limitation, we developed a compact AAV vector that enables the co-expression of Acidaminococcus sp. Cas12a (AsCpf1) and CRISPR RNAs (crRNAs) using a single bidirectional promoter derived from the mouse H1 promoter. Our single bidirectional H1 promoter supported indel formation comparable to that achieved by dual-promoter systems and facilitated scalable genome editing with single-, dual-, and triple-target configurations. Genome editing was successfully accomplished both in vitro and in vivo following AAV delivery. This study shows that our engineered compact AAV vector platform is capable of simultaneously delivering AsCpf1 and multiplexed crRNAs. Full article

Rice (Oryza sativa) production faces serious threats from multiple biotic stresses, particularly the brown planthopper, rice blast, and bacterial blight. Developing resistant cultivars is the most sustainable control strategy. Compared to race-specific resistance genes, disrupting susceptibility genes often confers broader and potentially more durable resistance. However, engineering broad-spectrum resistance against both insect pests and pathogens by editing susceptibility genes remains challenging. In this study, we employed multiplex CRISPR/Cas9 editing to simultaneously disrupt key susceptibility genes involved in distinct defense pathways: ACS2 (for brown planthopper), Bsr-D1, ERF922 or Pi21 (for fungal blast), and Xa5 (for bacterial blight). Three triple-mutant lines (abx, aex, and apx) were successfully generated, and all exhibited significantly enhanced resistance to brown planthopper, blast, and bacterial blight without compromising major agronomic traits compared to the wild type. Our work demonstrates the feasibility of multiplex susceptibility gene editing as a precise and efficient strategy for breeding rice varieties with synchronized, broad-spectrum resistance to both insect pests and pathogenic diseases. Full article

The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR–Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR–Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases. Full article

Background: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement. Methods: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2) and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed using the Agrobacterium-mediated method with the japonica rice variety Xin Dao 53 as the recipient. Mutation editing efficiency was detected in T₀ transgenic plants. Grain length, grain number per panicle, thousand-grain weight, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T₃ generations. Results: Effectively edited plants were obtained in the T₀ generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T₂ generation. In the T₃ generation, compared with the wild-type (WT), the edited homozygous lines showed increased grain number per panicle (14.60–25.61%), increased grain length (7.39–11.16%), increased grain length–width ratio (8.37–13.02%), increased thousand-grain weight (3.79–9.15%), a 42–64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits. Conclusions: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance. Full article

African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90–100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection. Full article

Indoor cat allergens, particularly the major allergen Fel d 1 protein, represent significant environmental triggers for allergic rhinitis, asthma, and other immune-related disorders in humans. With the continuous global increase in pet ownership, cat allergen proteins are prevalent in diverse settings and can even be transmitted to pet-free locations via clothing and animal fur, thereby posing health risks to sensitized individuals. This review systematically summarizes the molecular characteristics, distribution patterns, and mechanisms of human sensitization to indoor cat allergen proteins. It focuses on a comparative analysis of the principles, sensitivity, and application of commonly used immunological methods (such as various modified ELISAs, immunoblotting, and high-throughput multiplex detection technologies) alongside emerging real-time sensing platforms (including QCM, SAW, and LIF). Furthermore, this review summarizes key factors affecting indoor allergen concentrations, such as cat characteristics, architectural environments, human activities, and spatiotemporal variations. It also evaluates the efficacy and limitations of current allergy control strategies, covering source control (e.g., gene editing, immunomodulation), environmental management (e.g., air filtration), and medical treatments (e.g., allergen immunotherapy), and discusses future prospects. This review aims to offer a scientific foundation and systematic reference for the detection, control, and public health protection related to indoor cat allergens. Full article

In recent years, CRISPR-Cas9 technology has become a powerful and indispensable tool for targeted mutagenesis in plants, including applications such as gene knockout, prime-editing, multiplex gene editing, and regulation of gene transcription. As the number of potential genome editing approaches expands at a very fast pace, rapid, efficient, and cost-effective analytical strategies are needed to screen large numbers of mutants, including the detection of off-target events. In this study, we reported a detection method based on High-Resolution Melting (HRM) analysis to discriminate between wild-type (wt) and edited lines of different varieties of Vitis vinifera and grapevine rootstocks. Those edited lines were obtained through Agrobacterium tumefaciens mediated transformation of embryogenic calli using the CRISPR/SpCas9 system and targeting VviEPFL9-1 and VviEPFL9-2, two paralogous genes involved in stomata cell fate induction. The method clearly distinguished between the wt allele and the mutated one and was partially effective in distinguishing different types of mutation. Moreover, HRM data elaboration based on a Principal Component Analysis (PCA) allowed one to group populations of lines which originated from the same transformation event. Our study demonstrates the reliability of HRM as a fast and cost-effective diagnostic tool for the screening of edited lines and the evaluation of off-target events. Full article

Gene editing technologies, particularly CRISPR/Cas9, have revolutionized livestock genetics. They enable precise, efficient, and inheritable genome modifications. This review summarizes recent advances in the application of gene editing in livestock. We focus on six key areas: enhancement of disease resistance, improvement of growth performance and meat production traits, modification of milk composition, regulation of reproductive traits, adaptation to environmental stress, and promotion of animal welfare. For example, they have played an important role in improving mastitis resistance in cows, enhancing meat production performance in pigs, increasing milk yield in goats, and producing polled cows. Despite rapid progress, practical implementation in animal breeding still faces challenges. These include off-target effects, low embryo editing efficiency, delivery limitations, and ethical as well as regulatory constraints. Future directions emphasize the development of advanced editing tools, multiplex trait integration, and harmonized public policy. With continued innovation and responsible oversight, gene editing holds great promise for sustainable animal agriculture and global food security. Full article