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Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro Validation of sgRNA and Identifying Key Factors Influencing Efficiency.
CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an in vitro validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (Crassostrea gigas). We evaluated the efficiency of 11 sgRNAs targeting four genes both in vitro and in vivo in C. gigas. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in C. gigas, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.
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Non-genetically modified organism in vitro CRISPR/Cas9 gene editing of the lacZα gene: A 4.5 h laboratory course for senior high-school students.
The CRISPR/Cas9 system opens new horizons (M. Adli, Nat Commun, 2018) regarding genetic modifications of living organisms but also as an in vitro tool in laboratory protocols. Therefore, it boosts possibilities in research and future medical treatments. As the controversial claim of genomically edited babies by He Jiankui (Cyranoski D., Nature, 2019) demonstrates, the new gene editing potentials entail ethical discussions. A public or social discussion presupposes not only a theoretical knowledge or understanding of the system, but also profits from direct laboratory experiences showing how easy these techniques can be applied. Introducing numerous students and classes into these emerging techniques in a modern biology classroom depends on a suitable course concept, which fits legal and organizational requirements at the same time. Therefore, we implemented an appropriate hands-on laboratory course for senior high-school students, lasting just 4.5 h. Particularly with regard to European regulations concerning the handling of genetically modified organisms, the constructs and protocols avoid the transfer of Cas9 DNA. This normally mandatory transfer was replaced by in vitro gene-editing. This leads to Cas9 induced gene knock-outs due to frame shifts and/or the excision of DNA fragments in common Escherichia coli (E. coli) plasmids, such as pUC19. This gene knock-out concept covers various steps: In vitro plasmid editing with Cas9, ligation and transformation of E. coli cells with the modified plasmid DNA and finally the spread plating of transformed E. coli cells in order to analyze colonies after overnight incubation. The successful excision of DNA fragments by in vitro Cas9 treatment was determined by subsequent gel electrophoresis.
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CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis.
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CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.
Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.
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High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs.
Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.
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An LNP-CRISPR gene editing drug demonstrates efficacy and safety in patients with hereditary angioedema following in vivo administration.
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[Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system].
CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.
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The new normal of structure/function studies in the era of CRISPR/Cas9.
Major advances in gene-editing technologies have enabled the rapid dissection of proteins in complex biological systems, facilitating biological experiments to complement biochemical studies with purified components. In this editorial, we highlight CRISPR/Cas9-based strategies to rapidly manipulate endogenous genes - strategies that have already transformed functional studies of proteins in metazoan systems. We further describe emerging tools using a catalytically dead version of Cas9 (dCas9) that do not cleave DNA, but can alter gene expression and/or local chromatin states, edit single nucleotide bases, and permit the visualization of specific genomic loci. Looking to the not-too-distant future, CRISPR/Cas9-based methodologies promise to lead to discoveries of new biology, opening the door for bold new synthetic biology platforms.
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Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.
Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.
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All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.
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