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At least 19 recordsLinked to original sources

PubMed2026

CRISPR/Cas in gynecologic cancers: A review of experimental and therapeutic applications.

Gynecological malignancies-including cervical, ovarian, and endometrial cancers-remain a major global health challenge, contributing significantly to cancer-related morbidity and mortality among women. Despite advances in conventional treatments such as surgery, chemotherapy, radiotherapy, and immunotherapy, issues such as drug resistance, tumor recurrence, and limited efficacy in advanced-stage disease necessitate novel therapeutic strategies. The emergence of CRISPR/Cas-based genome editing has revolutionized cancer research by enabling precise, efficient, and programmable modifications of specific genomic loci. In gynecologic oncology, CRISPR/Cas systems have been employed to dissect oncogenic mechanisms, identify therapeutic targets, and develop innovative treatment modalities. In cervical cancer, CRISPR-mediated targeting of HPV E6 and E7 oncogenes has shown potential in restoring tumor suppressor pathways and enhancing chemosensitivity. In ovarian cancer, gene editing has been used to modulate chemoresistance, tumor angiogenesis, and metastasis through the knockout of key regulators such as DNMT1, EGFL6, and BRCA1/2. Similarly, in endometrial cancer, CRISPR tools have elucidated mechanisms of hormonal resistance and facilitated the development of in vivo models via somatic gene editing. This review highlights recent advances in the application of CRISPR/Cas technology to gynecologic malignancies, discussing its potential as both a therapeutic and research platform while acknowledging current limitations and translational hurdles.

Humans↗

PubMed2023

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Humans↗

PubMed2026

Emerging techniques of CRISPR/Cas system in antiviral therapy and diagnostics: Applications, limitations, and translational perspectives.

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.

CRISPR-Cas Systems↗

PubMed2026

Application of compact CRISPR/Cas nucleases for citrus genome editing.

Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.

CRISPR-Cas Systems↗

PubMed2025

In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.

Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.

Citrus↗

PubMed2026

SpacerScope: binary-vectorized, genome-wide off-target profiling for RNA-guided nucleases without prior candidate-site bias.

The precision of CRISPR/Cas systems is fundamental to their application in plant and animal biotechnology. However, comprehensive sequence-based off-target candidate discovery remains a computational bottleneck, particularly in large and complex genomes. Here we developed SpacerScope, an off-target candidate discovery framework that enables unbiased, genome-wide discovery by leveraging binary vectorization, bitwise filtering, and right-end-anchored alignment. Benchmarking against human CIRCLE-seq data demonstrated that SpacerScope recovered 100% of validated off-target sites (6142/6142), matching the sensitivity of exhaustive algorithms. Crucially, SpacerScope achieved this maximum candidate recovery while substantially reducing computational overhead. In large-genome evaluations, SpacerScope maintained low peak memory usage of 2.20 GiB and achieved substantial runtime improvements over indel-aware comparator tools, including more than 50-fold speedup relative to Cas-OFFinder 3 (544 s versus 29 185 s). Furthermore, comparative analyses in polyploid species, such as the octoploid strawberry, revealed that SpacerScope identified larger sequence-compatible candidate burdens than standard web-based design platforms. Our results establish SpacerScope as a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes. The source code and program was publicly available at https://github.com/charlesqu666/SpacerScope. Short Abstract CRISPR/Cas sequence-based off-target candidate discovery remains computationally challenging in large, repetitive, and polyploid genomes. Existing tools either miss indel-containing candidate sites or incur prohibitive runtime and memory costs. We developed SpacerScope, a binary-vectorized framework that enables unbiased, genome-wide off-target candidate discovery without pre-selected candidate sites. By integrating bitwise filtering with right-end-anchored alignment, SpacerScope recovered 100% of validated off-target sites in human CIRCLE-seq data while using only 2.20 GiB of memory and achieving more than 10-fold speedup over indel-aware alternatives. Evaluation in plant genomes, including rice and octoploid strawberry, further demonstrated SpacerScope's capacity to identify larger sequence-compatible candidate burdens overlooked by standard tools. SpacerScope thus provides a high-speed framework for sequence-based genome-wide off-target candidate discovery across diverse and highly repetitive genomic landscapes, supporting downstream prioritization.

CRISPR-Cas Systems↗

PubMed2026

Genome Editing in Solanaceae: Harnessing CRISPR-Cas Technology for Precision Crop Improvement.

Malnutrition and climate-induced stress remain major constraints to global food and nutritional security despite the yield gains of the Green Revolution. Solanaceae crops such as tomato, potato, brinjal, and pepper are key sources of vitamins, minerals, and bioactive compounds. Yet, their genetic improvement has been limited by narrow diversity and complex polygenic traits. The advent of CRISPR/Cas-mediated genome editing provides a transformative platform for precision crop improvement by enabling targeted modification of genes controlling stress tolerance, yield, and nutritional quality. In Solanaceae, CRISPR/Cas applications have successfully enhanced resistance against major pathogens (SlMlo1, SlPelo, SlDCL2), improved abiotic stress tolerance through editing of SlMAPK3, SlCBF1, and SlBZR1, and optimized fruit quality traits via modulation of Psy1, CrtR-b2, and fiAD2/3. Emerging innovations, such as base and prime editing, and RNP-mediated transgene-free delivery, are expanding the precision and scope of editing. However, challenges persist, including genotype-dependent transformation, low HDR efficiency, and incomplete understanding of off-target and epigenetic effects. Integrating CRISPR with omics-guided gene discovery, efficient transformation systems, and regulatory harmonization can accelerate the development of nutritionally enriched, stress-resilient, and sustainable Solanaceae varieties. This review synthesizes recent advances, identifies critical limitations, and outlines future opportunities for deploying CRISPR/Cas technology to achieve next-generation breeding and food system resilience.

CRISPR/Cas↗

PubMed2025

Enhanced cleavage of genomic CCR5 using CASX2Max.

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2Max in cellular gene excision. In sum, CasX2Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

Humans↗

PubMed2025

A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Bacteria↗

PubMed2025

CRISPR-Cas9 Genome Editing in Auxotrophic and Non-auxotrophic Fission Yeast Strains.

The CRISPR/Cas system is a very powerful genome-editing tool that has been developed over the past decade to optimize genome editing for many organisms. Here, we describe a rapid genome-editing method for fission yeast using the CRISPR-Cas9 system. It allows rapid generation of desired auxotrophic and non-auxotrophic strains without perturbing the local genome content by avoiding the insertion of selection markers at target loci.

CRISPR-Cas Systems↗

PubMed2026

Effects of CRISPR technology on agricultural sustainability: global applications and turkish perspective.

This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.

Plants, Genetically Modified↗

PubMed2024

Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.

Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.

Animals↗

PubMed2026

Gene therapy for genodermatoses at the crossroads of innovation and clinical translation.

Inherited genodermatoses are a heterogeneous group of rare monogenic disorders. Among these, epidermolysis bullosa (EB) and ichthyoses represent paradigmatic disorders characterized by severe skin fragility and hyperkeratosis, respectively, and impaired barrier function, often with profound effects on quality of life and systemic health. Current management remains largely palliative, underscoring the urgent need for disease-modifying therapies. Over the past 2 decades, advances in epithelial stem cell biology, vector engineering and genome editing technologies have transformed the therapeutic landscape for genodermatoses. Ex vivo gene therapy has provided the first proof that genetically corrected epidermal stem cells can achieve long-term tissue regeneration in EB skin patients, establishing a new paradigm for regenerative medicine. In parallel, the emergence of programmable genome engineering platforms, including CRISPR/Cas nucleases, base editors and prime editors, have enabled increasingly precise strategies for mutation-specific correction in both recessive and dominant disorders. Furthermore, the development of in vivo topical approaches is expanding the possibility of directly targeting the skin. Despite these advances, substantial translational barriers continue to limit broad clinical implementation. Efficient and durable targeting of epidermal stem cells within a highly regenerative tissue, together with safe delivery across the skin barrier, stringent control of off-target activity, scalable manufacturing and demonstration of long-term safety, remain major challenges for the clinical translation of these approaches. In this Review, we discuss the current state of gene therapy for genodermatoses, highlighting key clinical milestones, emerging genome editing technologies and next-generation delivery systems. We further examine the biological and regulatory challenges that need to be overcome to bridge the gap between experimental innovation and clinically accessible therapies for patients with inherited skin diseases.

epidermolysis bullosa (EB)↗

PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗

PubMed2026

From scissors to editors: how the evolution of precision is redefining therapeutic genome editing.

Since its introduction as a genome-editing tool, CRISPR-based technology has undergone rapid refinement, with precision emerging as a central focus of development. Early CRISPR-Cas9 systems demonstrated unprecedented ease and efficiency in targeting specific DNA sequences, but concerns over off-target effects and variable editing outcomes limited their broader application. This review outlines the progression of CRISPR from its discovery in prokaryotes to its application as a versatile tool in precision medicine, where it supports targeted therapies for genetic disorders in various ways. Although technical challenges, including off-target editing and delivery inefficiencies, persist alongside ethical considerations of accessibility and long-term consequences, CRISPR's ongoing refinements and innovations reflect a clear trajectory toward greater specificity, safety, and predictability, positioning CRISPR as an increasingly precise platform for both fundamental research and therapeutic use.

Gene Editing↗

PubMed2026

The application of the CRISPR-Cas system in Pseudomonas aeruginosa infections.

Due to the extensive drug resistance of Pseudomonas aeruginosa (P. aeruginosa), it is still a great clinical challenge. The clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) system has become a promising strategy against this pathogen. This review critically evaluates the multifaceted applications of CRISPR-Cas technology in P. aeruginosa, including its role in antimicrobial resistance, diagnostics, genome editing, and emerging therapeutic and vaccine strategies. In addition to conducting a comprehensive analysis of various studies, we also compared the performance and limitations of various CRISPR platforms, and discussed the main technologies and transformation obstacles in this field. Finally, we look forward to the direction of applying these experimental tools to clinical research in the future.

Pseudomonas aeruginosa↗

PubMed2026

Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.

Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.

Capsella↗

PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗