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Advanced gene editing techniques for enhancing disease resistance and climate resilience in crops.
Ensuring food security and solving the issues brought on by climate change require breeding and engineering of climate-resilient crops. Despite its contributions to reducing agricultural diseases, genetic engineering has several limitations, including high labor costs, lengthy processing times, and poor productivity. Genome editing has become a potential method to provide notable opportunities to explain complex biological processes, genetically solve the causes of diseases, and improve crops for disease resistance by effectively modifying multiple traits. Genome editing techniques including TALENs, ZFNs, and CRISPR/Cas9 increase agricultural productivity by developing climate-resistant crops and promoting climate-resilient agriculture. Among these approaches, CRISPR/Cas9 shows exceptional efficacy, minimal chance of off-target effects, and improved traits such as drought tolerance and disease resistance. This study explores advanced gene editing techniques for improving disease resistance in crops and developing climate-resilient varieties to reduce food insecurity and hunger. It demonstrates that these techniques have enhanced the nutritional content and resilience of many crops by fighting abiotic and biotic stresses. Future agricultural practices could alter the genes and improve disease-resistant crops by genome editing techniques.
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CRISPR/Cas: from adaptive immune system in prokaryotes to therapeutic weapon against immune-related diseases.
CRISPR/Cas evolved as an adaptive immune system in bacteria and archaea to inactivate foreign viral and plasmid DNA. However, the capacities of various CRISPR/Cas systems for precise genome editing based on sequence homology also allow their use as tools for genomic and epigenomic modification in eukaryotes. Indeed, these genetic characteristics have proven useful for disease modeling and testing the specific functions of target genes under pathological conditions. Moreover, recent studies provide compelling evidence that CRISPR/Cas systems could be useful therapeutic tools against human diseases, including cancer, monogenic disorders, and autoimmune disorders.HighlightsCRISPR/Cas evolved as an adaptive immune system in bacteria and archaea.CRISPR/Cas systems are nowadays used as tools for genomic modification.CRISPR/Cas systems could be useful therapeutic tools against human disease, including autoimmune conditions.
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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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High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters.
CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.
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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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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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User-Friendly Technology the Key to Gene-Editing's Bloom: Market for Gene-Editing Tools Estimated at $608m and Growing as New Applications Are Found.
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Gene Editing Provides Clues to Why Cancer Immunotherapy Often Fails.
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