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In Silico Design of gRNA for CRISPR/Cas9-Mediated Gene Knockout.
CRISPR/Cas9 stands as a revolutionary and versatile gene editing technology. At its core, the Cas9 DNA endonuclease is guided with precision by a specifically designed single-guide RNA (gRNA). This guidance system facilitates the introduction of double-stranded breaks (DSBs) within the DNA. Subsequent imprecise repairs, mainly through the non-homologous end-joining (NHEJ) pathway, yield insertions or deletions, resulting in frameshift mutations. These mutations are instrumental in achieving the successful knockout of the target gene. In this chapter, we describe all necessary steps to create and design a gRNA for a gene knockout to a target gene before to transfer it to a target plant.
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Gene Editing in Forest Tree Breeding for Stress Resistance: From Mechanisms to Future Prospects.
Forest ecosystems face escalating threats from climate change alongside a surging demand for sustainable bioproducts. While conventional tree breeding is inherently constrained by long generation cycles, high heterozygosity, and complex genomes, CRISPR-based genome editing provides a precision framework for targeted genetic improvement. This review synthesises the fundamental principles and limitations of multiple gene-editing technologies, with a particular emphasis on CRISPR systems (Cas9, Cas12, and Cas13), in the specific context of woody perennial biology. Recent applications in key forest genera, including Populus, Pinus, and Eucalyptus, demonstrate the efficacy of these gene-editing tools in manipulating complex traits, such as rewiring phytohormone signalling networks for drought tolerance or remodelling root system architecture to combat abiotic stress. We critically evaluate persistent translational bottlenecks in forest tree genome editing, with a specific focus on recalcitrant, genotype-dependent regeneration and the multifaceted challenges of long-term field validation. Finally, we highlight how synergising CRISPR technologies with multi-omics, genomic selection, and high-throughput phenomics can accelerate the development and application of climate-resilient woody perennials.
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CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.
In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.
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Performance enhancement of CRISPR-Cas system based on improved guide RNA: a review.
BACKGROUND: CRISPR-Cas technology has emerged as a transformative tool with widespread applications in gene editing and biosensing research; nevertheless, it is plagued by a suite of performance-related bottlenecks, including suboptimal targeting efficiency, undesirable off-target effects, insufficient sensitivity and recognition specificity, restricted target scope, limited multiplexing capacity, incompatible reaction systems, and compromised stability. As gRNA optimization has emerged as a core strategy to address these bottlenecks, there is an urgent need to consolidate recent breakthroughs in this rapidly advancing field. Existing literature lacks a comprehensive, focused synthesis of how gRNA optimization mitigates these key limitations, alongside an analysis of current challenges and future directions. RESULTS: Herein, this review comprehensively summarizes recent breakthroughs in augmenting CRISPR-Cas system performance through guide RNA (gRNA) optimization, and further dissects the current challenges, future prospects, and promising research directions in this rapidly advancing field. SIGNIFICANCE: It is timely to guide researchers in overcoming CRISPR-Cas performance barriers and accelerating its applications in gene editing and biosensing.
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Use of CRISPR/Cas9 to model brain diseases.
Aging-related brain diseases consist of a number of important neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases, all of which have become more prevalent as the life expectancy of humans is prolonged. Age-dependent brain disorders are associated with both environmental insults and genetic mutations. For those brain disorders that are inherited, gene editing is an important tool for establishing animal models to investigate the pathogenesis of disease and identify effective treatments. Here we focus on the tools for gene editing, especially CRISPR/Cas9, and discuss their application for generating animal models that can recapitulate the brain pathology seen in human diseases. We also highlight the advantages and disadvantages of establishing genetically modified animal models. Finally, we discuss recent findings to resolve technical issues related to the use of CRISPR/Cas9 for generating animal models of brain diseases.
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Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations.
We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.
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A guide for drug inducible genome editing with HIT systems.
Technologies toward precise control of biological events are desired for biomedical research and potential clinical applications. Our recently reported HIT systems based on CRISPR/Cas9 and TAL effectors can achieve temporal and dose dependent drug control for genome editing. Methods are presented for the application of these optimized HIT systems in human cells in this chapter.
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The history, use, and challenges of therapeutic somatic cell and germline gene editing.
The advent of directed gene-editing technologies now over 10 years ago ushered in a new era of precision medicine wherein specific disease-causing mutations can be corrected. In parallel with developing new gene-editing platforms, optimizing their efficiency and delivery has been remarkable. With their development, there has been interest in using gene-editing systems for correcting disease mutations in differentiated somatic cells ex vivo or in vivo or for germline gene editing in gametes or 1-cell embryos to potentially limit genetic diseases in the offspring and in future generations. This review details the development and history of the current gene-editing systems and the advantages and challenges in their use for somatic cell and germline gene editing.
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An efficient CRISPR/Cas9-mediated editing of phytoene desaturase in hexaploid sweetpotato.
CRISPR/Cas9-mediated gene editing has emerged as a pivotal tool for functional genomics and crop improvement. For the first time, we applied CRISPR/Cas9-mediated editing to the IbPDS gene in the purple-fleshed cultivar 'XZS-8', achieving mutation efficiencies up to 98.18 %. Loss-of-function mutations in IbPDS induced visually discernible albino phenotypes. Hi-TOM sequencing confirmed deletion mutations within the target locus, with editing efficiencies ranging from 17.77 % to 65.90 % (gRNA1) and 87.87-98.18 % (gRNA2). Knockout lines showed significant reductions in chlorophyll a and b content, confirming functional disruption of IbPDS. Collectively, our results demonstrate efficient CRISPR/Cas9-mediated genome editing for generating mutants in the hexaploid sweetpotato.
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Understanding the public's intention to adopt CRISPR-Cas9: the effect of beliefs, knowledge, and innovativeness.
CRISPR-Cas9 is a gene editing technology with wide-ranging medical potential and significant ethical implications. This study examined how personality traits, familiarity with CRISPR, knowledge about CRISPR, and beliefs concerning its applications are connected to the public’s willingness to adopt this technology, drawing on Rogers’ Diffusion of Innovations framework. A sample of 500 young adults aged 20–45 completed questionnaires assessing innovativeness as well as familiarity, knowledge, beliefs, and willingness to adopt CRISPR-Cas9 for therapeutic and non-therapeutic purposes. Results showed that only 24% of participants were familiar with CRISPR and that knowledge levels were generally low. Based on Rogers’ typology, 18.8% were identified as innovators, and 26.8% as early adopters, and only 3.2% as laggards. Beliefs were strongly associated with willingness to adopt CRISPR-Cas9 (r = .63, p < .001), moderating the weak associations of personality traits with adoption intentions. These findings suggest that beliefs are an important factor in CRISPR-Cas9 adoption and they appear to have more influence on willingness to adopt CRISPR-Cas9 than knowledge and innovativeness in this sample. Efforts to promote informed public discussion on CRISPR should be made, with an emphasis on ethical aspects alongside scientific information. This is critical for the responsible adoption of this technology.
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