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cRGD-modified hybrid lipopolymeric nanoplexes for gene editing in the posterior segment of the eye.
Eye-related diseases, specifically retinal dystrophy (RD) conditions, are the leading cause of blindness worldwide. Gene addition, regulation, or editing could potentially treat such diseases through gene expression regulation. CRISPR/Cas9 gene editing is one of the most prominent and precise gene editing tools which could be employed to edit genes related to the dystrophic condition. However, CRISPR/Cas9 faces in vivo delivery challenges due to its high molecular weight, negative charge, prone to degradation in the presence of nucleases and proteases, poor cellular degradation, etc., which makes it challenging to adopt for therapeutic applications. We developed cRGD-modified lipopolymeric nanoplexes loaded with Cas9 RNPs with a particle size and zeta potential of 175 ± 20 nm and 2.15 ± 0.9 mV, respectively. The cRGD-modified lipopolymeric nanoplexes were stable for 194 h and able to transfect >70 % ARPE-19 and NIH3T3 cells with an Indel frequency of ~40 % for the VEGF-A gene. The cRGD-modified lipopolymeric nanoplexes found good vitreous mobility and could transfection retinal cells in vivo after 48 h of intravitreal injection in Wistar Rats. Moreover, in vivo VEGFA gene editing was ~10 % with minimal toxicities. Collectively, the cRGD-modified lipopolymeric nanoplexes were found to have extreme potential in delivering CRISPR/Cas9 RNPs payload to the retinal tissues for therapeutic applications.
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Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.
Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.
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Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Therapeutics.
The recent progress in harnessing the efficient and precise method of DNA editing provided by CRISPR/Cas9 is one of the most promising major advances in the field of gene therapy. However, the development of safe and optimally efficient delivery systems for CRISPR/Cas9 elements capable of achieving specific targeting of gene therapy to the location of interest without off-target effects is a primary challenge for clinical therapeutics. Nanoparticles (NPs) provide a promising means to meet such challenges. In this review, we present the most recent advances in developing innovative NP-based delivery systems that efficiently deliver CRISPR/Cas9 constructs and maximize their effectiveness.
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CRISPR-Cas9 Based Bacteriophage Genome Editing.
Bacteriophages are the most abundant entities in the biosphere, and many genomes of rare and novel bacteriophages have been sequenced to date. However, bacteriophage functional genomics has been limited by a lack of effective research methods. Clustered regularly interspaced short palindromic repeat/CRISPR-associated gene (CRISPR-Cas) systems provide bacteriophages with a new mechanism for attacking host bacteria as well as new tools for study bacteriophage functional genomics. It has been reported that bacteriophages are not only the driving elements of the evolution of prokaryote CRISPR arrays but also the targets of CRISPR-Cas systems. In this study, a phage genome editing platform based on the heterologous CRISPR-Cas9 system was theoretically designed, and a Vibrio natriegens phage TT4P2 genome editing experiment was carried out in vivo in the host bacterium Vibrio natriegens TT4 to achieve phage gene deletion and replacement. The construction of this phage genome editing platform is expected to solve the problem of insufficient research on phage gene diversity, promote the development of phage synthetic biology and nanotechnology, and even accelerate the discovery of new molecular biology tools. IMPORTANCE Bacteriophages are the most numerous organisms on earth and are known for their diverse lifestyles. Since the discovery of bacteriophages, our knowledge of the wider biological world has undergone immense and unforeseen changes. A variety of V. natriegens phages have been detected, but few have been well characterized. CRISPR was first documented in Escherichia coli in 1987. It has been reported that the CRISPR-Cas system can target and cleave invaders, including bacteriophages, in a sequence-specific manner. Here, we show that the construction of a phage genome editing platform based on the heterologous CRISPR-Cas9 system can achieve V. natriegens phage TT4P2 gene editing and can also improve the efficiency and accuracy of phage TT4P2 gene editing.
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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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Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters.
Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.
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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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CRISPR deletions in cell lines for reconstitution studies of pseudokinase function.
The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.
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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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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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