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Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies.
Gene editing, particularly CRISPR/Cas technology, represents a promising approach for the treatment of rare genetic diseases, including inherited retinal dystrophies, for which effective therapies are largely unavailable. Despite extensive research investigating gene editing across a wide range of cell types, transient delivery of CRISPR/Cas components and efficient homology-directed repair (HDR) in differentiated cells remain challenging. In this study, we employed hiPSCs derived from patients with Stargardt disease or Best disease, carrying pathogenic variants in ABCA4 or BEST1, respectively, to explore gene editing in human models. CRISPR/Cas9 and Cas12 nucleases were delivered into hiPS-derived retinal pigment epithelium (RPE) and retinal organoids using lipoplexes and compared with electroporation. We evaluated transfection efficiency, sgRNA-mediated DNA cleavage, and HDR-based correction. Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction. Edited RPE cells preserved normal morphology and expressed specific maturity markers. In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues. This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes. These findings underscore the therapeutic potential of CRISPR/Cas-based strategies for inherited retinal dystrophies and provide a proof of concept for future clinical approximations.
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CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.
CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.
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Protocol to screen chemicals to enhance homology-directed repair in CRISPR-Cas9 gene editing.
CRISPR-Cas9-based gene editing via homology-directed repair (HDR) enables precise modifications, though its efficiency is limited by the prevalence of non-homologous end joining (NHEJ). Here, we present a protocol for enhancing HDR efficiency by identifying chemicals using high-throughput screening (HTS). We describe steps for designing 96-well plates, executing HTS, and performing data analysis. We then detail procedures for identifying small molecules that improve HDR-associated gene editing. This protocol has potential application in HTS analysis focused on discovering reliable HDR enhancers. For complete details on the use and execution of this protocol, please refer to Jang et al.1.
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A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.
CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
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Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species.
Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.
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Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.
CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene in melon. The constructed vectors were delivered to host plants using Agrobacterium-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a PDS editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.
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CRISPR/Cas9-Mediated Promoter Engineering in Saccharopolyspora erythraea.
In situ promoter engineering is an effective way to alter target gene expression without introducing excess DNA sequences. Recently, the CRISPR/Cas9 technologies have been proved to be efficient tools for genome editing in actinomycetes, making it easier and more efficient to perform gene insertion and substitution in actinomycetes in a scarless manner. In this chapter, we describe a routine protocol for CRISPR/Cas9-mediated promoter engineering in Saccharopolyspora erythraea NRRL 23338, which is the wild-type producer of erythromycin. This protocol can be adapted to CRISPR/Cas9-mediated gene editing, not limited to promoter engineering, in other actinomycetes, with modifications.
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Success in Sight for Gene Editing.
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Artificial Intelligence for Predictive Modeling in CRISPR/Cas9 Gene Editing: a Survey of Methods and Design Strategies.
Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.
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Aptazyme-mediated direct modulation of post-transcriptional sgRNA level for conditional genome editing and gene expression.
RNA-guided endonuclease Cas9 derived from microbial CRISPR-Cas adaptive immune systems is a powerful tool for genome editing, which has been widely used in eukaryotic systems, prokaryotic systems, and plants. However, the off-target effects caused by Cas9/sgRNA remain a major concern. Currently, the efforts to reduce the off-target effects mainly focus on improving the targeting specificity of sgRNA/Cas9, regulating the activity of the Cas9 protein or the sgRNA, and controlling the time window of their expression. In this study, a novel system was established to regulate the post-transcriptional sgRNA level by small molecule-controlled aptazyme. This system was shown to reduce the off-target effects caused by Cas9/sgRNA, while enabling precise temporal control over gene editing and regulatory activity. This new system could provide a potentially safer and more powerful tool for genome editing and therapeutic application.
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