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Cas9-Mediated Genome Engineering in Drosophila melanogaster.
The recent development of the CRISPR-Cas9 system for genome engineering has revolutionized our ability to modify the endogenous DNA sequence of many organisms, including Drosophila This system allows alteration of DNA sequences in situ with single base-pair precision and is now being used for a wide variety of applications. To use the CRISPR system effectively, various design parameters must be considered, including single guide RNA target site selection and identification of successful editing events. Here, we review recent advances in CRISPR methodology in Drosophila and introduce protocols for some of the more difficult aspects of CRISPR implementation: designing and generating CRISPR reagents and detecting indel mutations by high-resolution melt analysis.
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Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.
The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.
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Genome editing systems across yeast species.
Yeasts are used to produce a myriad of value-added compounds. Engineering yeasts into cost-efficient cell factories is greatly facilitated by the availability of genome editing tools. While traditional engineering techniques such as homologous recombination-based gene knockout and pathway integration continue to be widely used, novel genome editing systems including multiplexed approaches, bacteriophage integrases, CRISPR-Cas systems, and base editors are emerging as more powerful toolsets to accomplish rapid genome scale engineering and phenotype screening. In this review, we summarized the techniques which have been successfully implemented in model yeast Saccharomyces cerevisiae as well as non-conventional yeast species. The mechanisms and applications of various genome engineering systems are discussed and general guidelines to expand genome editing systems from S. cerevisiae to other yeast species are also highlighted.
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Revolutionizing cotton cultivation: A comprehensive review of genome editing technologies and their impact on breeding and production.
Cotton (Gossypium hirsutum L.), a vital global cash crop, significantly impacts both the agricultural and industrial sectors, providing essential fiber for textiles and valuable byproducts such as cottonseed oil and animal feed. The cultivation of cotton supports millions of livelihoods worldwide, particularly in developing regions, making it a cornerstone of rural economies. Despite its importance, cotton production faces numerous challenges, including biotic stresses from pests and diseases, and abiotic stresses like drought, salinity, and extreme temperatures. These challenges necessitate innovative solutions to ensure sustainable production. Genome editing technologies, particularly CRISPR/Cas9, have revolutionized cotton breeding by enabling precise genetic modifications. These advancements hold promise for developing cotton varieties with enhanced resistance to pests, diseases, and environmental stresses. Early genome editing tools like ZFNs and TALENs paved the way for more precise modifications but were limited by complexity and cost. The introduction of CRISPR/Cas-based technology with its simplicity and efficiency, has dramatically transformed the field, making it the preferred tool for genome editing in crops. Improved version of the technology like CRISPR/Cas12a, CRISPR/Cas13, base and prime editing, developed from CRISPR/Cas systems, provide additional tools with distinct mechanisms, further expanding their potential applications in crop improvement. This comprehensive review explores the impact of genome editing on cotton breeding and production. It discusses the technical challenges, including off-target effects and delivery methods for genome editing components, and highlights ongoing research efforts to overcome these hurdles. The review underscores the potential of genome editing technologies to revolutionize cotton cultivation, enhancing yield, quality, and resilience, ultimately contributing to a sustainable future for the cotton industry.
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Applications of genome editing on laboratory animals.
For four decades, genetically altered laboratory animals have provided invaluable information. Originally, genetic modifications were performed on only a few animal species, often chosen because of the ready accessibility of embryonic materials and short generation times. The methods were often slow, inefficient and expensive. In 2013, a new, extremely efficient technology, namely CRISPR/Cas9, not only made the production of genetically altered organisms faster and cheaper, but also opened it up to non-conventional laboratory animal species. CRISPR/Cas9 relies on a guide RNA as a 'location finder' to target DNA double strand breaks induced by the Cas9 enzyme. This is a prerequisite for non-homologous end joining repair to occur, an error prone mechanism often generating insertion or deletion of genetic material. If a DNA template is also provided, this can lead to homology directed repair, allowing precise insertions, deletions or substitutions. Due to its high efficiency in targeting DNA, CRISPR/Cas9-mediated genetic modification is now possible in virtually all animal species for which we have genome sequence data. Furthermore, modifications of Cas9 have led to more refined genetic alterations from targeted single base-pair mutations to epigenetic modifications. The latter offer altered gene expression without genome alteration. With this ever growing genetic toolbox, the number and range of genetically altered conventional and non-conventional laboratory animals with simple or complex genetic modifications is growing exponentially.
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UDiTaS™, a genome editing detection method for indels and genome rearrangements.
BACKGROUND: Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations. RESULTS: To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes. CONCLUSIONS: UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.
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CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.
BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.
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Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
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CRISPR Applications in Alzheimer's Disease: From High-Throughput Genetic Screening to Precision Editing and CNS Delivery.
Alzheimer's disease is a devastating progressive neurodegenerative disorder characterized by extracellular amyloid-beta plaques and intracellular tau tangles. Despite recent advancements in amyloid-beta-targeting immunotherapies, achieving safe and definitive disease control remains a profound clinical challenge. The CRISPR/Cas9 system has emerged as a powerful technology for precision neurogenetics, offering significant potential to address the fundamental questions behind Alzheimer's disease. This comprehensive review delineates the trajectory of CRISPR applications in Alzheimer's disease research and therapeutics. First, we explore the integration of CRISPR in engineering high-fidelity in vitro models, such as isogenic induced pluripotent stem cells and three-dimensional cerebral organoids, alongside advanced in vivo mammalian models. Second, we examine how these platforms facilitate unbiased high-throughput genetic screening to uncover molecular underpinnings regulating tau, lipid metabolism, and neuroinflammation. Third, we critically evaluate precision editing strategies targeting core risk genes (APP, MAPT, APOE, and TREM2), explicitly highlighting the severe physiopathological trade-offs between therapeutic efficacy and loss-of-function toxicity. Finally, we address the ultimate translational bottlenecks impeding clinical application. By dissecting the packaging limits of adeno-associated viral vectors and the physical barricade of the blood-brain barrier, we underscore the necessity of transitioning toward next-generation base editors and non-viral lipid nanoparticles to realize safe and efficacious in vivo clinical gene therapies against Alzheimer's disease.
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RNAi and genome editing of sugarcane: Progress and prospects.
Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan-genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods-biolistic gene transfer or Agrobacterium-mediated transformation-coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture-free technologies, such as spray-induced gene silencing, virus-induced gene silencing, or virus-induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.
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