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Found: 26 168
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Breathing new life into in vivo lung editing.
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CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. Reply.
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A CRISPR/Cas9 system adapted for gene editing in marine algae.
Here we report that the CRISPR/Cas9 technology can be used to efficiently generate stable targeted gene mutations in microalgae, using the marine diatom Phaeodactylum tricornutum as a model species. Our vector design opens for rapid and easy adaption of the construct to the target chosen. To screen for CRISPR/Cas9 mutants we employed high resolution melting based PCR assays, mutants were confirmed by sequencing and further validated by functional analyses.
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High-throughput sequencing revealed low-efficacy genome editing using Cas9 RNPs electroporation and single-celled microinjection provided an alternative to deliver CRISPR reagents into Euglena gracilis.
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CRISPR/Cas9 in soybean research: a measured leap toward genetic refinement.
Soybean [Glycine max (Linn.) Merr] is a globally important source of plant-based protein and edible oil, widely cultivated in India as a major oilseed crop. However, conventional breeding approaches for soybean are laborious, constrained by the genomic complexity of soybean, and face persistent challenges in genetic transformation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based genome editing has emerged as a precise and efficient tool for rapid trait improvement in soybean. While many existing reviews of soybean have been published, this literature review addresses some fundamental challenges with respect to genome editing. This study summarizes recent advancements in soybean genome editing, offering insights into the practical informatics of soybean-specific genomic databases that are required for successful construct design. It also discusses emerging technologies such as base editing, prime editing, and the increasingly important role of artificial intelligence in accelerating target identification and construct design. It highlights applications in nutritional enhancement, abiotic and biotic stress tolerance, and improvement of key agronomic traits. Beyond the conventional topics, it addresses the current regulatory landscape that researchers must navigate for releasing genome-edited varieties and provides a comprehensive perspective on practical solutions to overcome major constraints in soybean genetic engineering.
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Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells.
Genetically modified HSV-1 viruses serve as promising vectors for tumour therapy and vaccine development. The CRISPR/Cas9 system is one of the most powerful tools for precise gene editing of the genomes of organisms. However, whether the CRISPR/Cas9 system can precisely and efficiently make gene replacements in the genome of HSV-1 remains essentially unknown. Here, we reported CRISPR/Cas9-mediated editing of the HSV-1 genome in human cells, including the knockout and replacement of large genes. In established cells stably expressing CRISPR/Cas9, gRNA in coordination with Cas9 could direct a precise cleavage within a pre-defined target region, and foreign genes were successfully used to replace the target gene seamlessly by HDR-mediated gene replacement. Introducing the NHEJ inhibitor SCR7 to the CRISPR/Cas9 system greatly facilitated HDR-mediated gene replacement in the HSV-1 genome. We provided the first genetic evidence that two copies of the ICP0 gene in different locations on the same HSV-1 genome could be simultaneously modified with high efficiency and with no off-target modifications. We also developed a revolutionized isolation platform for desired recombinant viruses using single-cell sorting. Together, our work provides a significantly improved method for targeted editing of DNA viruses, which will facilitate the development of anti-cancer oncolytic viruses and vaccines.
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SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs.
Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.
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The State-of-the-Art of Gene Editing and its Application to Viral Infections and Diseases Including COVID-19.
Gene therapy delivers a promising hope to cure many diseases and defects. The discovery of gene-editing technology fueled the world with valuable tools that have been employed in various domains of science, medicine, and biotechnology. Multiple means of gene editing have been established, including CRISPR/Cas, ZFNs, and TALENs. These strategies are believed to help understand the biological mechanisms of disease progression. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been designated the causative virus for coronavirus disease 2019 (COVID-19) that emerged at the end of 2019. This viral infection is a highly pathogenic and transmissible disease that caused a public health pandemic. As gene editing tools have shown great success in multiple scientific and medical areas, they could eventually contribute to discovering novel therapeutic and diagnostic strategies to battle the COVID-19 pandemic disease. This review aims to briefly highlight the history and some of the recent advancements of gene editing technologies. After that, we will describe various biological features of the CRISPR-Cas9 system and its diverse implications in treating different infectious diseases, both viral and non-viral. Finally, we will present current and future advancements in combating COVID-19 with a potential contribution of the CRISPR system as an antiviral modality in this battle.
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Rogues and Regulation of Germline Editing.
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A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.
Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.
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