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In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
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CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations.
The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9's clinical efficacy, positioning them as pivotal tools for future gene-editing therapies.
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Gene-Targeted DNA Methylation: Towards Long-Lasting Reprogramming of Gene Expression?
DNA methylation is an essential epigenetic mark, strongly associated with gene expression regulation. Aberrant DNA methylation patterns underlie various diseases and efforts to intervene with DNA methylation signatures are of great clinical interest. Technological developments to target writers or erasers of DNA methylation to specific genomic loci by epigenetic editing resulted in successful gene expression modulation, also in in vivo models. Application of epigenetic editing in human health could have a huge impact, but clinical translation is still challenging. Despite successes for a wide variety of genes, not all genes mitotically maintain their (de)methylation signatures after editing, and reprogramming requires further understanding of chromatin context-dependency. In addition, difficulties of current delivery systems and off-target effects are hurdles to be tackled. The present review describes findings towards effective and sustained DNA (de)methylation by epigenetic editing and discusses the need for multi-effector approaches to achieve highly efficient long-lasting reprogramming.
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A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent.
Bacillus velezensis (Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (loc, srf, bl, and fen) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.
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Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.
Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.
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Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.
Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.
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Nanoneedle Array-Electroporation Facilitates Intranuclear Ribonucleoprotein Delivery and High Throughput Gene Editing.
Dendritic cells (DCs) are critical regulators of T cell immunity, with immense therapeutic potential against tumors and autoimmune diseases. Efficient gene editing in DCs is crucial for understanding their regulatory mechanisms and maximizing their therapeutic efficacy. However, DCs are notoriously difficult to transfect, posing a major bottleneck for conventional DNA and RNA-based editing approaches. Microneedle-mediated injection of Cas9/sgRNA ribonucleoprotein (RNP) directly into the nucleus, akin to gene editing in reproductive cells, offers promise but suffers from limitations in scalability. Here, an intranuclear delivery system using a hollow nanoneedle array (HNA) combined with nano-electroporation is developed. The 2 µm-high HNA physically reaches the nucleus, positioning the nuclear envelope and plasma membrane in close proximity at the tip. Transient electronic pulses then induce simultaneous perforations across all 3 membranes, enabling direct RNP delivery into the nucleus. This HNA-based system achieves efficient knockout of genes like PD-L1 in primary DCs, demonstrating its potential as a powerful tool for gene editing in DCs and other hard-to-transfect cells.
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A synthetic guide RNA scaffold enhanced CRISPR/Cas9 editing efficiency in plants across multiple gene targets.
CRISPR/Cas9 mediated genome editing is a highly powerful and versatile tool for accelerating crop improvement. The editing efficiency of CRISPR/Cas9 system in planta has been highly variable owing to the variable binding affinity between native CRISPR RNA and Cas9 protein in vivo. In plant systems, systematic, large-scale engineering and benchmarking of guide RNA (gRNA) scaffold variants is still relatively limited compared with work in mammalian systems, despite several important studies demonstrating that scaffold and expression-cassette engineering can substantially improve CRISPR/Cas9 efficacy. Current study addresses the limitations of commonly used gRNA scaffold architecture by incorporating a stabilized stem-loop RAR (tetra loop) extension and a transcription-termination site mutation, resulting in improved RNA folding, increased Cas9 binding affinity, and enhanced in vivo editing outcomes. The synthesized scaffold boosted CRISPR/Cas9 efficiency in monocot or dicot plants across the 19 diverse target sites in Arabidopsis, rice and tomato. Furthermore, the synthetic scaffold is compatible with multiplex genome editing architectures, including polycistronic tRNA-gRNA (PTG) expression systems, enabling efficient simultaneous targeting of multiple genomic loci. The findings of this study have broad applications in precision plant breeding, functional genomics, and agricultural biotechnology, facilitating reliable gene modification across diverse plant species and transformation platforms.
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Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production.
The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.
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TALEDs complete the toolkit for editing human mitochondrial DNA.
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