Drosophila melanogaster is a highly versatile model organism that has profoundly advanced our understanding of human diseases. With more than 60% of its genes having human homologs, Drosophila provides an invaluable system for modelling a wide range of pathologies, including neurodegenerative disorders, cancer, metabolic diseases, as well as cardiac and muscular conditions. This review highlights key developments in utilizing Drosophila for disease modelling, emphasizing the genetic tools that have transformed research in this field. Technologies such as the GAL4/UAS system, RNA interference (RNAi) and CRISPR-Cas9 have enabled precise genetic manipulation, with CRISPR-Cas9 allowing for the introduction of human disease mutations into orthologous Drosophila genes. These approaches have yielded critical insights into disease mechanisms, identified novel therapeutic targets and facilitated both drug screening and toxicological studies. Articles were selected based on their relevance, impact and contribution to the field, with a particular focus on studies offering innovative perspectives on disease mechanisms or therapeutic strategies. Our findings emphasize the central role of Drosophila in studying complex human diseases, underscoring its genetic similarities to humans and its effectiveness in modelling conditions such as Alzheimer's disease, Parkinson's disease and cancer. This review reaffirms Drosophila's critical role as a model organism, highlighting its potential to drive future research and therapeutic advancements.

The recent approval of the first gene editing therapy for sickle cell disease and transfusion-dependent beta-thalassemia by the U.S. Food and Drug Administration (FDA) demonstrates the immense potential of CRISPR (clustered regularly interspaced short palindromic repeats) technologies to treat patients with genetic disorders that were previously considered incurable. While significant advancements have been made with ex vivo gene editing approaches, the development of in vivo CRISPR/Cas gene editing therapies has not progressed as rapidly due to significant challenges in achieving highly efficient and specific in vivo delivery. Adeno-associated viral (AAV) vectors have shown great promise in clinical trials as vehicles for delivering therapeutic transgenes and other cargos but currently face multiple limitations for effective delivery of gene editing machineries. This review elucidates these challenges and highlights the latest engineering strategies aimed at improving the efficiency, specificity, and safety profiles of AAV-packaged CRISPR/Cas systems (AAV-CRISPR) to enhance their clinical utility.

Neurotensin (Nts) is a peptide that acts via neurotensin receptors and is implicated in multiple aspects of physiology and behavior, including modulating feeding and body weight. How and where the Nts signaling system mediates these effects, and via which of its receptor isoforms is incompletely understood. This review examines the role of Nts signaling via the periphery and central nervous system on feeding and body weight. These data highlight various ways in which the Nts system contributes to feeding and body weight that differ depending on the site, tissue, and the Nts or Nts receptor-expressing cell type in question. Given that the Nts system does not convey the same signaling throughout the body, constitutive approaches modulating the expression or signaling of the Nts signaling system may not provide sufficient resolution to reveal how it impacts feeding. Combining neuropharmacology and site-specific approaches holds promise define the broad range of mechanisms by the Nts system modulates feeding and satiety and its contributions to normal and disrupted feeding states.

Base editors (BEs) are a promising tool for precise base conversion in human cells and animals, while the adeno-associated virus (AAV) is the major vector for human gene therapy. However, the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector, making dual-AAV approaches based on trans-splicing intein necessary. Even with this approach, current split DNA cassettes are still larger than the AAV packing limit, posing a challenge for cellular production of AAV. Moreover, some split strategies yield variable editing results and target coverage. To address these limitations, 25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively, with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide. Fortunately, the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb. These novel dual-AAV systems, designated 4.6AAV-CBE and 4.7AAV-ABE, were found to have base editing efficiencies similar to wild-type BEs, with a narrower editing window than the current 573 split system. Notably, 4.6AAV-CBE yield a higher AAV production titer, up to 2.1-fold in AAV-N and 1.5-fold in AAV-C, compared to the split-573BE system, likely due to the reduction of DNA cassette size within the AAV packaging capacity. Moreover, after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells, the multiplicities of infection (MOI) and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system. This study present advanced dual-AAV systems for ABE and CBE delivery, establishing a basis for safe and efficient BE therapies.

In situ imaging diagnosis and precise treatment of deep tumor tissues are hotspots in life sciences and medical research. In recent years, using focused ultrasound to remotely control engineered bacteria for drug release has become one of the methods for precise in vivo drug delivery. However, non-visualized engineered bacteria pose challenges for precise control within the body. Therefore, there is an urgent need for an engineered bacterial vector capable of deep tissue imaging to precisely locate bacteria in vivo. Acoustic reporter genes (ARGs) are biological elements used for deep tissue imaging, with gene clusters over 8 kb. However, ARGs are often tested on plasmids, which hinders stable expression in vivo and limits the space for inserting components that regulate drug release. Therefore, we used the attenuated Salmonella typhimurium VNP20009, known for its tumor-targeting ability, as the chassis bacteria. By using CRISPR-Cas9 technology, we inserted ARGs into the genome and optimized the promoter strength and copy number for ARG expression, constructing ultrasound-visible engineered bacteria expressing gas vesicles on the genome. Additionally, by knocking out the stress protein gene htrA in VNP20009, we increased the maximum injection dose by tenfold and the tumor specificity by a hundredfold. The constructed ultrasound-visible engineered bacteria can stably synthesize gas vesicles and output ultrasound signals while directly carrying drug plasmids for tumor therapy. Our research provides an effective vector for diagnosis and precise treatment.

Antimicrobial resistance (AMR) poses a growing global threat. This review examines AMR from diverse angles, tracing the story of antibiotic resistance from its origins to today's crisis. It explores the rise of AMR, from its historical roots to the urgent need to counter this escalating menace. The review explores antibiotic classes, mechanisms, resistance profiles, and genetics. It details bacterial resistance mechanisms with illustrative examples. Multidrug-resistant bacteria spotlight AMR's resilience. Modern AMR control offers hope through precision medicine, stewardship, combination therapy, surveillance, and international cooperation. Converging traditional and innovative treatments presents an exciting frontier as novel compounds seek to enhance antibiotic efficacy. This review calls for global unity and proactive engagement to address AMR collectively, emphasizing the quest for innovative solutions and responsible antibiotic use. It underscores the interconnectedness of science, responsibility, and action in combatting AMR. Humanity faces a choice between antibiotic efficacy and obsolescence. The call is clear: unite, innovate, and prevail against AMR.

Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.

R-loops, three-strand nucleic acid structures, have emerged as crucial players in various physiological processes, including the regulation of gene expression, DNA replication, and class switch recombination. However, their presence also poses a significant threat to genome stability. A particularly challenging aspect is understanding the dynamic balance between R-loops' "light" and "dark" sites, especially concerning maintaining genome integrity. The complex and multifaceted roles of R-loops in genome stability necessitate a deeper understanding. This review offers a comprehensive exploration of the formation, resolution, and implications of R-loops, particularly in the context of DNA damage and human disease. We delve into the dualistic nature of R-loops, highlighting their role in DNA damage response and repair, and discuss the therapeutic potential arising from our evolving understanding of these enigmatic entities. Emphasizing recent advancements and unresolved questions, this review aims to provide a cohesive overview of R-loops, inviting further inquiry and investigation into their complex biological significance.

CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates-containing a mismatch in the region complementary to RNA-by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a-crRNA complex and compared it with Cas9-sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a-crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9-sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.

Filamentous fungi are essential industrial microorganisms that can serve as sources of enzymes, organic acids, terpenoids, and other bioactive compounds with significant applications in food, medicine, and agriculture. However, the underdevelopment of gene editing tools limits the full exploitation of filamentous fungi, which still present numerous untapped potential applications. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system, a versatile genome-editing tool, has advanced significantly and been widely applied in filamentous fungi, showcasing considerable research potential. This review examines the development and mechanisms of genome-editing tools in filamentous fungi, and contrasts the CRISPR/Cas9 and CRISPR/Cpf1 systems. The transformation and delivery strategies of the CRISPR/Cas system in filamentous fungi are also examined. Additionally, recent applications of CRISPR/Cas systems in filamentous fungi are summarized, such as gene disruption, base editing, and gene regulation. Strategies to enhance editing efficiency and reduce off-target effects are also highlighted, with the aim of providing insights for the future construction and optimization of CRISPR/Cas systems in filamentous fungi.

Nucleic acid drugs represent the latest generation of precision therapeutics, holding significant promise for the treatment of a wide range of intractable diseases. Delivery technology is crucial for the clinical application of nucleic acid drugs. However, extrahepatic delivery of nucleic acid drugs remains a significant challenge. Systemic administration often fails to achieve sufficient drug enrichment in target tissues. Localized administration has emerged as the predominant approach to facilitate extrahepatic delivery. While localized administration can significantly enhance drug accumulation at the injection sites, nucleic acid drugs still face biological barriers in reaching the target lesions. This review focuses on non-viral nucleic acid drug delivery techniques utilized in local administration for the treatment of extrahepatic diseases. First, the classification of nucleic acid drugs is described. Second, the current major non-viral delivery technologies for nucleic acid drugs are discussed. Third, the bio-barriers, administration approaches, and recent research advances in the local delivery of nucleic acid drugs for treating lung, brain, eye, skin, joint, and heart-related diseases are highlighted. Finally, the challenges associated with the localized therapeutic application of nucleic acid drugs are addressed.

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in retinal degeneration 12 (rd12) of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

Developments in basic stem cell biology have paved the way for technology translation in human medicine. An exciting prospective use of stem cells is the ex vivo generation of hepatic and pancreatic endocrine cells for biomedical applications. This includes creating novel models 'in a dish' and developing therapeutic strategies for complex diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetes. In this review, we explore recent advances in the generation of stem cell-derived hepatocyte-like cells and insulin-producing β-like cells. We cover the different differentiation strategies, new discoveries, and the caveats that still exist regarding their routine use. Finally, we discuss the challenges and limitations of stem cell-derived therapies as a clinical strategy to manage metabolic diseases in humans.

The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.

Modification of sgRNA has been considered as a necessary approach to enhance the stability and cleavage efficiency of the CRISPR/Cas9 system. In this study, a rigid G-quadruplex structure was genetically applied to the 3' end of typical sgRNA for protection of RNA from 3'-5' exoribonuclease degradation. The in vitro transcriptional production yields of sgRNAs bearing G-quadruplex structure such as sgRNA3 and sgRNA4 were around 1.4 and 1.5 times higher than the yield of typical sgRNA1, respectively. The results have also shown that appending G-quadruplex motif at the 3' end of typical sgRNAs did minorly affect the cleavage activity of CRISPR/Cas9. Interestingly, cleavage efficiency of CRISPR/Cas9 system with sgRNAs bearing the rigid G-quadruplex was fully retained in the presence of 3'-5' exoribonucleases such as RNase II or RNase R. In contrast, the cleavage activity of CRISPR/Cas9 system with the typical sgRNA1 was significantly decreased in the same condition. This protection of sgRNA through G-quadruplex structure-based modifications might provide a potential approach for improving cleavage efficiency of CRISPR/Cas9 system in the exoribonuclease environment.

The online version contains supplementary material available at 10.1007/s13205-025-04354-x.

Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.

Sex determination pathways regulate male and female-specific development and differentiation and offer potential targets for genetic pest management methods. Insect sex determination pathways are comprised of primary signals, relay genes and terminal genes. Primary signals of coleopteran, dipteran, hymenopteran and lepidopteran species are highly diverse and regulate the sex-specific splicing of relay genes based on the primary signal dosage, amino acid composition or the interaction with paternally inherited genes. In coleopterans, hymenopterans and some dipterans, relay genes are Transformer orthologs from the serine-arginine protein family that regulate sex-specific splicing of the terminal genes. Alternative genes regulate the splicing of the terminal genes in dipterans that lack Transformer orthologs and lepidopterans. Doublesex and Fruitless orthologs are the terminal genes. Doublesex and Fruitless orthologs are highly conserved zinc-finger proteins that regulate the expression of downstream proteins influencing physical traits and courtship behaviours in a sex-specific manner. Genetic pest management methods can use different mechanisms to exploit or disrupt female-specific regions of different sex determination genes. Female-specific regions of sex determination genes can be exploited to produce a lethal gene only in females or disrupted to impede female development or fertility. Reducing the number of fertile females in pest populations creates a male-biased sex ratio and eventually leads to the local elimination of the pest population. Knowledge on the genetic basis of sex determination is important to enable these sex determination pathways to be exploited for genetic pest management.

The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.

This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings.

CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms.

CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.

The possibility of editing the genomes of human embryos has generated significant discussion and interest as a matter of science and ethics. While it holds significant promise to prevent or treat disease, research on and potential clinical applications of human embryo editing also raise ethical, regulatory, and safety concerns. This systematic review included 223 publications to identify the ethical arguments, reasons, and concerns that have been offered for and against the editing of human embryos using CRISPR-Cas9 technology. We identified six major themes: risk/harm; potential benefit; oversight; informed consent; justice, equity, and other social considerations; and eugenics. We explore these themes and provide an overview and analysis of the critical points in the current literature.

Fowl adenovirus (FAdV) poses incessant outbreaks to poultry production worldwide, and Inclusion body hepatitis (IBH) is a predominant FAdV infectious disease. Currently, limited vaccines are available in Malaysia to fight against the local predominant FAdV strain 8b isolate (FAdV-8b), posing a desperate demand for efficient vaccine development. The fiber protein of FAdV is one of the major constituents of the adenoviral capsid involved in the virulence of pathogens. Hence, the aim was to modify the fiber gene of FAdV-8b UPMT27 to develop a live attenuated FAdV vaccine via the gene-editing CRISPR/Cas9 technology. Primary specific pathogen-free (SPF) chicken embryonic liver cells (CELs) infected with the modified isolated (cfUPMT27) were reported with significantly reduced cytopathic effects, delayed viral localization into the nucleus, and low apoptotic rates. cfUPMT27 isolate also exhibited constant amino acid substitution of Y179D in subsequent passages. Meanwhile, the liver of cfUPMT27 inoculated-SPF chicken embryonic eggs (CEE) was observed with mild hydropericardium and reported with a delayed mortality at 6-days post-infection (dpi). This holistic, integrative study incorporating genetic, pathology, and immunology analysis proposed cfUPMT27 isolate as a candidate vaccine for FAdV infections, providing efficient future protection in chickens.

Growing world population and deteriorating climate conditions necessitate the development of new crops with high yields and resilience. CRISPR-Cas-mediated genome engineering presents unparalleled opportunities to engineer crop varieties cheaper, easier and faster than ever. In this Review, we discuss how the CRISPR-Cas toolbox has rapidly expanded from Cas9 and Cas12 to include different Cas orthologues and engineered variants. We present various CRISPR-Cas-based methods, including base editing and prime editing, which are used for precise genome, epigenome and transcriptome engineering, and methods used to deliver the genome editors into plants, such as bacterial-mediated and viral-mediated transformation. We then discuss how promoter editing and chromosome engineering are used in crop breeding for trait engineering and fixation, and important applications of CRISPR-Cas in crop improvement, such as de novo domestication and enhancing tolerance to abiotic stresses. We conclude with discussing future prospects of plant genome engineering.

Helicobacter pylori possesses an intrabacterial nanotransportation system (ibNoTS) for transporting VacA, CagA, and urease within the bacterial cytoplasm. This system is controlled by the extrabacterial environment. The transport routes of the system for VacA have not yet been studied in detail. In this study, we demonstrated by immunoelectron microscopy that VacA localizes closely with the MreB filament in the bacterium, and the MreB polymerization inhibitor A22 obstructs the transport of VacA by ibNoTS. These findings indicate that the route of ibNoTS for VacA is closely associated with the MreB filament Additionally, it was confirmed that VacA does not closely associate with the bacterial filament FtsZ, which is involved in the transport of the virulence factor urease, as previously suggested. We propose that the route of ibNoTS for VacA is associated with the MreB filament in H. pylori.

Pre-eclampsia (PE) is a serious pregnancy disorder linked to genetic factors, particularly the ACVR2A gene, which encodes a receptor involved in the activin signaling pathway and plays a critical role in reproductive processes. Transcriptomic data analysis and experimental verification confirmed a downregulation of ACVR2A expression in placental tissues from PE patients. In this study, CRISPR/Cas9 technology was employed to investigate the effect of ACVR2A gene deletion on trophoblast cells using the HTR8/SVneo and JAR cell lines. Deletion of ACVR2A inhibits trophoblastic migration, proliferation, and invasion, underscoring its pivotal role in cellular function. RNA-seq data analysis unveiled an intricate regulatory network influenced by ACVR2A gene knockout, especially in the TCF7/c-JUN pathway. By employing RT-PCR and immunohistochemical analysis, a potential association between ACVR2A and the TCF7/c-JUN pathway was hypothesized and confirmed. The complexity of PE onset and the significance of genetic factors were emphasized, particularly the role of the ACVR2A gene identified in genome-wide association study. This study established a robust foundation for delving deeper into the intricate mechanisms of PE, paving the way for focused early intervention, personalized treatment, and enhanced obstetric healthcare.

CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage parametrized by data from the literature, including single-molecule Förster resonance energy transfer (FRET) measurements. Based on observed FRET transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds.

Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding, independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference effects based on the target position and strand. Genome-wide transcriptome profiling in single-guide RNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology.

Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.

CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of its active conformation with a full R-loop. Capitalizing on recent advances in Cas9 structural determination, we constructed a catalytic-state Cas9 model bound to a bona fide R-loop and performed an integrated computational investigation. Our molecular dynamics simulations reveal substantial conformational heterogeneity in the PAM (protospacer-adjacent motif)-distal nontarget DNA strand and adjacent Cas9 regions, leading to dynamically fluctuating interactions, thereby challenging experimental resolution of the full R-loop complex. Comparative analysis highlights a conformational barrier restricting final activation of the HNH nuclease domain, suggesting that strategic modulation of HNH interactions on its two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics simulations indicate that with H983 protonated at Nε, the RuvC domain favors a phosphate-mediated over a histidine-mediated pathway for nontarget strand cleavage. Additionally, we identify an alternative HNH-mediated target strand cleavage pathway, involving a water nucleophile aligned at the 5' side of the scissile phosphate. Inspired by the basic residue ladder observed in RuvC, we propose extending a similar ladder in HNH to strengthen DNA binding and catalytic activity. Our study provides critical insights into Cas9 structure, dynamics, and catalysis, laying a foundation for the rational design of next-generation CRISPR-Cas9 systems with optimized specificity-efficiency balance.

Recent advancements in synthetic biology and sequencing technologies have revolutionized the ability to manipulate viral genomes with unparalleled precision. This review focuses on two powerful methodologies: deep mutational scanning and CRISPR-based genome editing, that enable comprehensive mutagenesis and detailed functional characterization of viral proteins. These approaches have significantly deepened our understanding of the molecular determinants driving viral evolution and adaptation. Furthermore, we discuss how these advances provide transformative insights for future vaccine development and therapeutic strategies.

Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.

We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.

Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.

Artificial DNA nanostructures, with their sequence programmability, precise molecular recognition and tunable stimuli responsiveness, bridge material chemistry and biomedicine. Here we detail the design and construction of hybridization chain reaction (HCR)-based DNA nanoframeworks, a class of DNA nanostructures with programmable sequences and customizable functions. HCR is an efficient, enzyme-free amplification strategy that isothermally produces nicked double-stranded DNA with periodically repeated modules via the assembly of two DNA hairpins, triggered by a DNA initiator. In contrast to other available assembly methods for the synthesis of DNA nanostructures, such as tile-mediated assembly, DNA origami and rolling circle amplification, the HCR method offers improved stability and efficiency under mild conditions, without reliance on enzymatic activity. The procedure uses radical polymerization to integrate DNA initiator into nanoframeworks, with overhangs complementary to functional sequences - termed linkers -which are amplified and incorporated through HCR. The linkers enable the incorporation of functional nucleic acid sequences. The HCR-based DNA nanoframeworks facilitate the loading capability of the delivered molecules, showing notable therapeutic efficacy and biosensing sensitivity. Preparation time for HCR-based DNA nanoframeworks ranges from 30 h to 45 h, depending on the payload.

Theranostic platforms that integrate diagnostic and therapeutic functionalities offer promising strategies for precision medicine, particularly in the treatment of major diseases. However, the development of platforms capable of achieving spatially controlled detection and therapy at the lesion site remains a significant challenge. Herein, we present a dual-stimuli-responsive DNA nanoframework that achieve spatially controlled codelivery of molecular beacon (MB) and Cas9 ribonucleoprotein (RNP), enabling simultaneous specific optical detection and efficient gene therapy for pancreatic cancer. The DNA nanoframeworks are synthesized via precipitation polymerization, utilizing acrylamide-modified DNA to initiate a hybridization chain reaction that facilitates the effective loading of MB-extended and sgRNA-conjugated DNA hairpins. The Cas9 protein is efficiently loaded into the nanoframeworks through phase transition-induced polymer chain rearrangement, overcoming steric hindrance. Upon aptamer-mediated internalization into PANC-1 cells, the overexpressed apurinic/apyrimidinic endonuclease 1 and ribonuclease H in cancer cells induce site-specific cleave of MB and DNA-RNA hybrid duplex, respectively. This cleavage restores fluorescence for specific optical detection, whereas the released Cas9 RNP performs gene editing for efficient therapy. Low fluorescence background and favorable biocompatibility are observed in normal cells. In a pancreatic cancer mouse model, the platform demonstrates significant detection-guided antitumor efficacy, highlighting its potential for precision medicine in cancer therapy.

Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.

The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.

Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.

With the global aging trend, neurodegenerative diseases (NDs) have emerged as a significant public health concern in the 21st century, imposing substantial economic burdens on families and society. NDs are characterized by cognitive and motor decline, resulting from a combination of genetic and environmental factors. Currently, there is no cure for NDs. Gene and RNA editing therapies offer new possibilities for addressing NDs. Gene editing involves modifying mutant genes associated with NDs, while RNA editing can directly modify RNA molecules to regulate the protein translation process, potentially influencing the expression of genes related to NDs. In this review, we examined the historical evolution, mechanisms of action, applications in NDs, advantages and disadvantages, as well as ethical and safety considerations of gene and RNA editing. While gene and RNA editing technologies hold promise for treating NDs, further research and development are needed to address safety, efficacy, and treatment timing issues, ultimately offering improved treatment options for ND patients. Our review provides valuable insights for future gene and RNA editing applications in ND treatment.

CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.

This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.

Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.

Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.

Limonene is a chiral, high-demand monoterpene that has wide applications in therapeutics, cosmetics, biofuels, agri-food, biomaterials, and solvent industries. However, its biosynthesis by microbial cell factories is often limited by the poor activity of limonene synthase (LS). Optimization of the rate-limiting enzyme is thus crucial for boosting limonene production. Here, we report the identification of ten LS homologues from sequence data mining and their testing in cells accumulating geranyl pyrophosphate (GPP) or neryl pyrophosphate (NPP) for limonene production. The selectivity of these enzymes toward GPP or NPP was investigated, leading to the identification of a limonene synthase from Agastache rugosa that displays a clear substrate preference for NPP over GPP in vivo. This enzyme was selected as a template for engineering. Using in silico analyses and mutagenesis, several mutants were engineered that revealed differences in substrate specificity. Among them, a combination of mutations (S8K/I265V/E276P/P277R/A281K/N282T/I285Q/I286L) improved limonene production by 4.8- and 1.9-fold with the GPP and NPP pathways, respectively. The mutant predominantly produced (+)-limonene from GPP and a mixture of limonene from NPP, with ∼85-90% of (+)-limonene. This decreased the selectivity for NPP by 2.4-fold. This supports the improved biological production of limonene enantiomers from renewable carbon sources.

Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.

CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)-rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine-cytosine (GC)-rich PAMs, have remained inaccessible. This study develops a small RNA toxin-aided strategy to evolve ErCas12a for targeting GC-rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC-rich PAMs and target five times more PAM sequences than the wild-type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non-canonical PAMs, including GC-rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non-canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.