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Found: 26 147
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Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system.
Among CRISPR-Cas genome editing systems, Streptococcus pyogenes Cas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probiotic Lactobacillus rhamnosus. We have confirmed the predicted 5'-NGAAA-3' PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9's A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond.
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Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.
CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.
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[Precision gene editing technologies based on CRISPR/Cas9: a review].
Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.
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Novel genome-editing-based approaches to treat motor neuron diseases: Promises and challenges.
Motor neuron diseases are untreatable with common pharmacological approaches. Spinal muscular atrophy (SMA) is caused by SMN1 gene mutations leading to lowered SMN expression. Symptoms are alleviated in infants with a higher copy number of the SMN2 gene, which, however, displays a splicing defect resulting in low SMN levels. Amyotrophic lateral sclerosis (ALS) is caused by a number of mutations, with C9orf72 repeat expansions the most common genetic cause and SOD1 gain-of-function mutations the first genetic cause identified for this disease. Genetic therapies based on oligonucleotides that enhance SMN2 splicing and SMN production or lower SOD1 expression have shown promise in initial clinical trials for individuals with SMA and ALS harboring SOD1 mutations, respectively. Gene addition/silencing approaches using adeno-associated viruses (AAVs) are also currently under clinical investigation in trials for SMA and ALS. Here we provide a brief overview of these efforts and their advantages and challenges. We also review genome editing approaches aimed at correcting the disease-causing mutations or modulating the expression of genetic modifiers, e.g., by repairing SOD1 mutations or the SMN2 splicing defect or deleting C9orf72 expanded repeats. These studies have shown promising results to approach therapeutic trials that should significantly lower the progression of these deadly disorders.
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TargeTron Technology Applicable in Solventogenic Clostridia: Revisiting 12 Years' Advances.
Clostridium has great potential in industrial application and medical research. But low DNA repair capacity and plasmids transformation efficiency severely delay development and application of genetic tools based on homologous recombination (HR). TargeTron is a gene editing technique dependent on the mobility of group II introns, rather than homologous recombination, which makes it very suitable for gene disruption of Clostridium. The application of TargeTron technology in solventogenic Clostridium is academically reported in 2007 and this tool has been introduced in various clostridia as it is easy to operate, time saving, and reliable. TargeTron has made great progress in solventogenic Clostridium in the aspects of acetone-butanol-ethanol (ABE) fermentation pathway modification, important functional genes identification, and xylose metabolic pathway analysis and reconstruction. In the review, 12 years' advances of TargeTron technology applicable in solventogenic Clostridium, including its principle, technical characteristics, application, and efforts to expand its capabilities, or to avoid potential drawbacks, are revisisted. Some other technologies as putative competitors or collaborators are also discussed. It is believed that TargeTron combined with CRISPR/Cas-assisted gene/base editing and gene-expression regulation system will make a better future for clostridial genetic modification.
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Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.
Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.
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Advancing chloroplast bioengineering: Innovations, regulatory challenges, and translational pathways for sustainable agriculture.
Escalating climate instability and rising global food demand necessitate the development of resilient crop systems underpinned by precise, predictable, and rapidly deployable genetic innovations. Chloroplast bioengineering has emerged as a vanguard strategy, offering a uniquely tractable platform characterized by the organelle's distinct plastome, discrete copy number, and predominantly maternal inheritance. Recent advances in plastid transformation and base editing now enable the high-fidelity, multiplex introduction of photosynthetic, osmoprotective, and redox-regulating pathways across diverse plant lineages. Controlled-environment and field-proximal trials demonstrate that chloroplast-engineered metabolic modules enhance CO2 assimilation, stabilize photochemistry under heat and drought stress, and improve osmotic buffering capacity. Beyond trait improvement, chloroplasts function as high-capacity production organelles capable of accumulating oral or mucosal biologics at levels that reduce reliance on cold-chain logistics. However, challenges such as species-level recalcitrance, prolonged timelines for achieving homoplasmy, and limited scalability of current plastid biomanufacturing pipelines continue to constrain broad agricultural and biopharmaceutical deployment. To overcome these barriers, we propose a standardized, empirically testable framework integrating optimized transformation workflows, quantitative trait benchmarking, multi-location field validation, and techno-economic analyses. This framework embeds long-term stewardship principles, including marker-free selection, proactive resistance management, and FAIR-aligned data transparency, while emphasizing equitable access pathways for resource-limited regions. Collectively, this work positions chloroplast engineering as a mechanistically grounded, field-ready platform poised to reshape crop resilience, sustainable bioproduction, and global biologic accessibility amid accelerating climate stress.
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Precise DNA base editing using AlphaFold3-based contact modelling.
Achieving high specificity in biochemical transformations is crucial for research and therapeutics. This is particularly important for genome editing, where enhancing tool specificity ensures effective and precise editing outcomes1,2. Current strategies are constrained by activity-specificity trade-offs, high labour intensity and low success rates3,4. Here we present ContactSeek, an artificial-intelligence-driven framework that uses AlphaFold3 (AF3)-predicted contact probability5 to improve the specificity of genome editors. Using Cas9-TadA adenine base editors6-8 as a demonstration, we mapped their genome-wide off-targets and fed the off-target DNA sequences to AF3. Among AF3 outputs, we found that contact probability was more sensitive than predicted three-dimensional structures for detecting differential interactions between on- and off-target complexes. Correlating contact probability with sequencing-based off-target signals, ContactSeek identified and ranked consensus contact regions, which are neighbouring Cas residues with consistent contact changes to DNA/guide RNA, and pinpointed specificity-determining residues within them. ContactSeek can also be applied modularly and identified key residues in the TadA8e deaminase. Targeted amplicon sequencing, genome-wide profiling, R-loop assay and RNA-sequencing together confirmed the greatly enhanced specificity; our best variant, combining two mutations of Cas9 and TadA8e, outperformed several known high-fidelity adenine base editors. ContactSeek is also generalized to Cas12a-based cytosine base editors. Collectively, our framework represents an AF3-driven model tailored for specificity improvement, establishing a paradigm for improving the precision of genome editing tools through the integration of structural and functional dimensions.
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In Utero Gene Therapy for Sickle Cell Disease: Current Evidence, Ethical Considerations, and Future Directions-A Scoping Review.
PURPOSE: Sickle Cell Disease (SCD) is a monogenic, autosomal recessive disorder caused by mutations in the β-globin gene, resulting in the production of abnormal hemoglobin S (HbS). When deoxygenated HbS polymerizes, it causes red blood cells to become sickle-shaped, and prone to hemolysis. These abnormal and sickled cells obstruct small blood vessels, leading to vaso-occlusion and tissue ischemia, contributing to multiple complications. SCD impacts nearly 8 million people worldwide, and is estimated to affect 500,000 newborns annually. Historically, potentially curative therapies have been limited, but advances in fetal diagnosis and gene-editing technologies have highlighted the potential use of in utero gene therapy as a strategy to correct the β-globin mutation before clinical disease onset. This scoping review summarizes the current evidence on in utero and fetal gene-editing strategies for SCD. METHODS: A systematic search of PubMed, Embase, and Scopus was conducted from database inception through September 2025 following PRISMA-ScR guidelines. Eligible studies included preclinical and translational investigations, early clinical reports, and expert policy or consensus statements focusing on in utero gene therapy or fetal gene editing for SCD. 25 studies met inclusion criteria and were categorized into 4 domains: ex vivo or in vivo gene editing, in utero gene editing, transplantation and tolerance, and reviews/policy/framework. FINDINGS: Preclinical studies demonstrated that targeted in utero gene editing can achieve postnatal correction of disease-relevant alleles in animal models using CRISPR-Cas9 and base-editing technologies. Lipid nanoparticle (LNP) and viral vector delivery platforms enabled efficient editing of fetal hematopoietic stem cells, supporting long-term persistence of edited genes. Transplantation studies showed that fetal immune tolerance can persist as well. Review and consensus statements emphasized ethical, regulatory, and procedural considerations, including maternal autonomy, risk assessment, and long-term monitoring. Collectively, the evidence supports the feasibility of prenatal intervention as an acceptable and potentially curative approach for SCD. IMPLICATIONS: In utero gene therapy represents a promising strategy for early correction of the β-globin mutation, with the potential to prevent disease onset and reduce long-term morbidity. However, translation to clinical practice will require additional studies and nuanced ethical oversight. Future research should prioritize the optimal timing of intervention, maternal-fetal immune compatibility, and equitable access to ensure accessible clinical implementation.
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Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation.
The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications.
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