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Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae.
Bioethanol, as a form of renewable and clean energy, has become increasingly important to the energy supply. One major obstacle in ethanol production is developing a high-capacity system. Existing approaches for regulating the ethanol production pathway are relatively insufficient, with nonspecific genetic manipulation. Here, we used CRISPR/Cas9 technology to disrupt the alcohol dehydrogenase (ADH) 2 gene via complete deletion of the gene and introduction of a frameshift mutation in the ADH2 locus. Sequencing demonstrated the accurate knockout of the target gene with 91.4% and near 100% targeting efficiency. We also utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single-guide RNAs. This extensive analysis indicated the mutations in the CRISPR/Cas9-engineered strains were homozygous. We applied the engineered Saccharomyces cerevisiae strains for bioethanol production. Results showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain. This work illustrates the applicability of this highly efficient and specific genome engineering approach to promote the improvement of bioethanol production in S. cerevisiae via metabolic engineering. Importantly, this study is the first report of the disruption of a target gene, ADH2, in S. cerevisiae using CRISPR/Cas9 technology to improve bioethanol yield.
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Bioethics Panels Open Door Slightly to Germline Gene Editing.
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Gene editing in legal limbo in Europe.
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Enhancing genome editing efficiency in goldfish (Carassius auratus) through utilization of CRISPR-Cas12a (Cpf1) temperature dependency.
The CRISPR/Cas technology has demonstrated revolutionary potential across various fields, including agriculture, medicine, and food safety detection. However, the utility of CRISPR/Cas12a, a particularly promising gene-editing tool, is constrained by its temperature sensitivity, limiting its application in low-temperature environments. In this study, we developed a gene-editing technique based on the CRISPR/Cas12a system in the poikilothermic species goldfish Carassius auratus. We systematically evaluated the editing efficiencies of LbCas12a and AsCas12a on the tyrosinase (tyr) gene under varying temperature conditions. Our results revealed a pronounced temperature dependence of Cas12a, with elevated temperatures markedly enhancing its editing activity, particularly for AsCas12a. A brief one-hour high-temperature treatment was sufficient to achieve effective gene disruption, underscoring CRISPR/Cas12a as a rapid and efficient gene-editing tool. Temperature was utilized as a conditional trigger for Cas12a-mediated gene knockout, enabling precise modulation of gene disruption at specific embryonic developmental stages. Whole-genome resequencing of the mutants confirmed the absence of off-target effects, further emphasizing the precision of this editing process. These findings indicated that CRISPR/Cas12a represented a viable alternative to the widely utilized CRISPR/Cas9 system and could be applied in conjunction, thereby expanding the potential applications of gene-editing technologies.
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Gene editing advances on all fronts.
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Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.
Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe3+ with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.
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[A new milestone in human embryo editing].
Viable human embryos carrying a disease mutation have now been corrected by CRISPR-Cas9 editing. Although the correction is not perfect, and the embryos have not been implanted, this brings us closer to human germline editing and to the associated ethical questions.
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Genome Editing of Monkey.
Gene-modified monkey models would be particularly valuable in biomedical and neuroscience research. Virus-based transgenic and programmable nucleases-based site-specific gene editing methods (TALEN, CRISPR-cas9) enable the generation of gene-modified monkeys with gain or loss of function of specific genes. Here, we describe the generation of transgenic and knock-out (KO) monkeys with high efficiency by lentivirus and programmable nucleases.
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An Accessible Intersectional Transgenic Single-Vector CRISPR/Cas9 Platform for Precise Gene Editing and Functional Analysis.
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CRISPR-Cas Gene Editing to the Genetic Rescue.
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