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RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells.
In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.
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A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis.
Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.
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Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.
Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.
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Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.
CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.
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High-efficiency prime editing with optimized, paired pegRNAs in plants.
Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.
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Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System.
Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.
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In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease.
In vivo gene editing in post-mitotic neurons of the adult brain may be a useful strategy for treating neurological diseases. Here, we develop CRISPR-Cas9 nanocomplexes and show they were effective in the adult mouse brain, with minimal off-target effects. Using this system to target Bace1 suppressed amyloid beta (Aβ)-associated pathologies and cognitive deficits in two mouse models of Alzheimer's disease. These results broaden the potential application of CRISPR-Cas9 systems to neurodegenerative diseases.
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The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.
Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.
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CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use.
For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR-Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don't exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.
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Dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma.
Choroidal melanoma is the most common primary intraocular malignancy, and effective gene therapy remains limited by ocular barriers, tumor heterogeneity, and inefficient nucleic acid delivery. This review critically analyses the molecular rationale, engineering principles, emerging nanoarchitectures, biological validation, and translational prospects of dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma. The manuscript also highlights key challenges and future strategies for clinical implementation. Current evidence indicates that dual-vector platforms enable independent optimization of mRNA and CRISPR cargos while maintaining coordinated spatiotemporal therapeutic activity. We conducted a literature search in PubMed, Scopus, and Web of Science for relevant studies published from January 2015 to September 2026. Functional cargo partitioning, vector modularity, programmable intracellular trafficking, and ocular-targeted nanoengineering improve delivery precision and reduce biological compromise associated with co-encapsulation. Lipid, polymeric, hybrid, biomimetic, and viral-nonviral architectures each provide distinct engineering advantages depending on therapeutic objectives. Preclinical studies support enhanced genome editing, transient protein restoration, improved tumor suppression, and reduced off-target effects. Nevertheless, manufacturing scalability, regulatory standardization, long-term safety, and clinical validation remain major barriers to translation. Dual-vector nanoengineered mRNA-CRISPR systems represent a promising next-generation platform for precision gene therapy in choroidal melanoma by enabling coordinated and programmable therapeutic delivery. Continued advances in modular nanoengineering, personalized cargo selection, and clinically scalable manufacturing will be essential for successful translation into precision ocular oncology.
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