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Effect of BRD0539 on Gene Editing and Mosaicism Rate in Porcine Gene Editing Embryos by CRISPR/Cas9.
Microinjection is a common method for generating gene-edited animals; however, persistent Cas9 activity post-cleavage often results in mosaic embryos due to editing occurring in different blastomeres. This study investigated whether co-injecting the CRISPR/Cas9 system with the small-molecule Cas9 inhibitor BRD0539, or supplementing it in the culture medium, could reduce mosaicism while maintaining editing efficiency in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. The findings are as follows: Co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA: sgRNA significantly reduced gene editing efficiency (28.5% ± 11.6% and 33.8% ± 4.1%, respectively, vs. 86.9% ± 4.5% in control, p < 0.05). Supplementing the culture medium with 10 or 50 μM BRD0539 also reduced both editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6%, respectively, vs. 85.6% ± 4.8%) and mosaicism rate (25.0% ± 15.9% and 12.5% ± 12.5%, respectively, vs. 87.1% ± 7.8%, p < 0.05). Immunofluorescence revealed sustained Cas9 protein expression up to 48 h post-injection. Crucially, short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation significantly reduced mosaicism (32.6% ± 7.5% vs. 78.7% ± 9.6%, p < 0.05) without compromising editing efficiency. Furthermore, this treatment did not adversely affect cleavage rates, blastocyst development, total cell number. These results demonstrate that transient inhibition of Cas9 activity with 10 μM BRD0539 during a critical window effectively reduces mosaicism in microinjected porcine embryos, offering a promising strategy to enhance the efficiency of generating non-mosaic gene-edited livestock.
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Highly efficient CRISPR-mediated gene editing in a rotifer.
Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.
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Application of CRISPR/Cas9-Mediated Gene Editing in Tomato.
CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.
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Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus.
Portunus trituberculatus is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.
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Human Genome Editing and a Global Socio-bioethics Approach.
A global socio-bioethics is called upon to address the ethical challenges arising from the revolutionary gene editing technologies such as CRISPR-Cas9, which offers the capability to rewrite the human genome. The ethical inquiry Françoise Baylis has undertaken in the book Altered Inheritance: CRISPR and the Ethics of Human Genome Editing (Harvard University Press, 2019) operates at individual, societal and global levels. Baylis has not only presented insights on how to practice "slow science" and achieve broad societal consensus through empowering the public, but she also shown what a global socio-bioethics approach can offer for the further development of bioethics.
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Gene-editing pipeline takes off.
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Antibody-mediated delivery of CRISPR-Cas9 ribonucleoproteins in human cells.
The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.
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CRISPR/Cas-Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen-Resistant Pulse Crops.
Pulses provide myriad health benefits and are advantageous in an environmental context as a result of their leguminous nature. However, phytopathogenic fungi, oomycetes and bacteria pose a substantial threat to pulse production, at times leading to crop failure. Unfortunately, existing disease management strategies often provide insufficient control, and there is a clear need for the development of new pulse cultivars with durable and broad-spectrum disease resistance. CRISPR/Cas-mediated gene editing has proven its potential for rapidly enhancing disease resistance in many plant species. However, this tool has only very recently been applied in pulse species, and never in the context of plant immunity. In this review, we examine the recent successful utilization of this technology in pulse species for proof-of-concept or the improvement of other traits. In addition, we consider various genes that have been edited in other plant species to reduce susceptibility to pathogens, and discuss current knowledge regarding their roles in pulses. Given the functional conservation of the selected genes across diverse plant species, there is a high likelihood that their editing would elicit similar effects in non-oilseed grain legumes, thus providing a suite of potential targets for CRISPR/Cas-mediated gene editing to promote pulse crop productivity in coming years.
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Pre-clinical non-viral vectors exploited for in vivo CRISPR/Cas9 gene editing: an overview.
Clustered regulatory interspaced short palindromic repeats or CRISPR/Cas9 has emerged as a potent and versatile tool for efficient genome editing. This technology has been exploited for several applications including disease modelling, cell therapy, diagnosis, and treatment of many diseases including cancer. The in vivo application of CRISPR/Cas9 is hindered by poor stability, pharmacokinetic profile, and the limited ability of the CRISPR payloads to cross biological barriers. Although viral vectors have been implemented as delivery tools for efficient in vivo gene editing, their application is associated with high immunogenicity and toxicity, limiting their clinical translation. Hence, there is a need to explore new delivery methods that can guarantee safe and efficient delivery of the CRISPR/Cas9 components to target cells. In this review, we first provide a brief history and principles of nuclease-mediated gene editing, we then focus on the different CRISPR/Cas9 formats outlining their potentials and limitations. Finally, we discuss the alternative non-viral delivery strategies currently adopted for in vivo CRISPR/Cas9 gene editing.
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Strategies for the Production and Phenotypic Analysis of Mutations in the Mouse.
The laboratory mouse is one of the primary model organisms for human biology and genetics. The production and phenotypic analysis of mutations in the mouse has made great strides with targeted mutagenesis in embryonic stem (ES) cells and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene editing in preimplantation embryos. Mutations in many genes are now available or can be easily produced. For anyone planning to use mutational analysis for a research program, this article introduces an overall strategy for obtaining or generating a mutation and systematically analyzing the phenotype.
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