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[Control (editing) of the genome within reach, or already in our hands?].
Although different genome editing tools have been around for decades, the recent emergence of cheap, quick, and accessible CRISPR/Cas9 technology has led to a revolution in this field. The technique has the potential to transform medicine from curative into preventive using a gene therapy. An application of genome editing has proven to be effective for both genetic and non-genetic (e.g. infectious) diseases. However, cancer and rare diseases treatment is at the forefront of interest. Concurrently, the legal and ethical frameworks should be discussed, especially as the technology moves towards a modification of the germ cells or embryos. In addition to a precise molecular genetic diagnosis and choosing the best gene therapy approach for a particular individual, an attention should also be paid to the impacts on the entire human population and the next generations. In this review, we summarize the most important applications of CRISPR/Cas9 technology in the field of medicine. Some interesting results from recent years are presented in the context of used approaches and importance for the future developments in medicine. Finally, ethical and legal conditions in relation to different gene editing applications are discussed.
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Gene and epigenetic editing in the treatment of primary ciliopathies.
Primary ciliopathies are inherited human disorders that arise from mutations in ciliary genes. They represent a spectrum of severe, incurable phenotypes, differentially involving several organs, including the kidney and the eye. The development of gene-based therapies is opening up new avenues for the treatment of ciliopathies. Particularly attractive is the possibility of correcting in situ the causative genetic mutation, or pathological epigenetic changes, through the use of gene editing tools. Due to their versatility and efficacy, CRISPR/Cas-based systems represent the most promising gene editing toolkit for clinical applications. However, delivery and specificity issues have so far held back the translatability of CRISPR/Cas-based therapies into clinical practice, especially where systemic administration is required. The eye, with its characteristics of high accessibility and compartmentalization, represents an ideal target for in situ gene correction. Indeed, studies for the evaluation of a CRISPR/Cas-based therapy for in vivo gene correction to treat a retinal ciliopathy have reached the clinical stage. Further technological advances may be required for the development of in vivo CRISPR-based treatments for the kidney. We discuss here the possibilities and the challenges associated to the implementation of CRISPR/Cas-based therapies for the treatment of primary ciliopathies with renal and retinal phenotypes.
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Improvements in algal lipid production: a systems biology and gene editing approach.
BACKGROUND: In the wake of rising energy demands, microalgae have emerged as potential sources of sustainable and renewable carbon-neutral fuels, such as bio-hydrogen and bio-oil. PURPOSE: For rational metabolic engineering, the elucidation of metabolic pathways in fine detail and their manipulation according to requirements is the key to exploiting the use of microalgae. Emergence of site-specific nucleases have revolutionized applied research leading to biotechnological gains. Genome engineering as well as modulation of the endogenous genome with high precision using CRISPR systems is being gradually employed in microalgal research. Further, to optimize and produce better algal platforms, use of systems biology network analysis and integration of omics data is required. This review discusses two important approaches: systems biology and gene editing strategies used on microalgal systems with a focus on biofuel production and sustainable solutions. It also emphasizes that the integration of such systems would contribute and compliment applied research on microalgae. CONCLUSIONS: Recent advances in microalgae are discussed, including systems biology, gene editing approaches in lipid bio-synthesis, and antenna engineering. Lastly, it has been attempted here to showcase how CRISPR/Cas systems are a better editing tool than existing techniques that can be utilized for gene modulation and engineering during biofuel production.
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Pronuclear Microinjection of One-Cell Embryos.
Generating a transgenic or gene-modified mouse requires the introduction of exogenous reagents into an early-stage embryo. The mouse one-cell embryo or zygote possesses two pronuclei, representing the genetic contribution of the sperm and oocyte. Traditional transgenic mice are generated by injecting a DNA solution containing a purified transgene construct into the male pronucleus, generally the larger of the two pronuclei. Similarly, gene-editing reagents such as ZFNs, TALENs, and CRISPR RNAs are introduced into zygotes in the same manner, making this technique applicable to a wide variety of projects. This chapter presents the procedures for pronuclear microinjection.
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CRISPR-Based Therapy for Ischemic Stroke: A Narrative Review.
Ischemic stroke (IS) is one of the most common neurological diseases worldwide and is caused by the blockage of cerebral blood vessels, leading to reduced blood flow and neuronal damage. Given the limitations of existing treatments, CRISPR gene-editing technology has emerged as a promising strategy to precisely target the molecular pathways underlying IS pathophysiology. By enabling intervention in genes regulating inflammation, apoptosis, and repair, CRISPR enables more precise and effective therapies. Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells. Studies have shown that the use of CRISPR-Cas9 to modulate key pathogenic pathways, including those governing inflammation, oxidative stress, and cell death, can prevent neuronal damage and improve neurological function. Additionally, targeting ncRNAs and RNA methylation with CRISPR-based systems plays a role in regulating oxidative stress and stress granule formation. The use of CRISPR to modulate cell communication and organelle transfer and correct mitochondrial mutations has also been considered a neuroprotective mechanism. Despite persistent challenges in targeted and safe delivery, substantial preclinical advances, primarily in rodent models, underscore the potential for CRISPR-based therapies to transform future stroke treatment. These findings suggest that CRISPR-based strategies could evolve into precision neurotherapeutics that address root molecular pathologies, potentially complementing or surpassing current stroke interventions.
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Application of CRISPR-Cas9 based gene editing to study the pathogenesis of colon and liver cancer using organoids.
Two breakthrough techniques that have totally revolutionized biology in last 1 decade are the discovery of genome editing tools and growing the stem cells/primary tissue explants in defined 3D culture. In this regard the discovery of CRISPR-Cas9 as a specific gene editing tool and organoid culture from adult stem cell has provided easy handy tools to uncover the process of organ development and also modeling cancer. Genetically modified organoids have been developed by sequential knockout and knockin of driver mutations by genome editing followed by niche-based selection. The modified organoids when xenotransplanted in animal models faithfully recapitulate the neoplastic events of human tumors. The present review focuses on the merging of these two powerful technologies in understanding the complexities of colon and liver cancer.
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Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.
OBJECTIVE: Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection. RESULTS: Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.
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Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing.
To explore the editing specificity of CRISPR/Cpf1 system, effects of target mutation were systematically examined using a reporter activation assay, with a set of single-nucleotide mutated target site. Consistent with our previous study performed with CRISPR/Cas9, a "core" sequence region that is highly sensitive to target mutation was characterized. The region is of 4-nucleotide long, located from +4 to +7 position of the target site, and positioned within a positively charged central channel when assembled into Cpf1 endonuclease. Single-nucleotide mutation at the core sequence could abolish gene editing mediated by a however active sgRNA. With a great majority of the target sites, a kind of 'super' off-target gene editing was observed with both CRISPR/Cpf1 and CRISPR/Cas9. For a given target site, mutation at certain positions led to greatly enhanced off-target gene editing efficacy, even up to 10-fold of that of the fully-matched target. Study further found that these effects were determined by the identity of target nucleotide, rather than the nucleotide of crRNA. This likely suggests that the interactions between target nucleotide and the endonuclease are involved in this process.
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The emerging patent landscape of CRISPR-Cas gene editing technology.
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One-in-one individual package and delivery of CRISPR/Cas9 ribonucleoprotein using apoferritin.
So far, most reported delivery of CRISPR/Cas9 is achieved by internalized or encapsulated multiple ribonucleoprotein units into only one carrier unit, with relatively large size. Here, we report a novel, small-sized, individual package of CRISPR/Cas9, via using tetralysine modified H-chian apoferritin (TL-HFn) as packaging material. In this paper, each CRISPR/Cas9 complex is proved to be successfully installed into one TL-HFn (~26 nm), and delivered into the targeting cell via TfR1-mediated endocytosis. We found that after 6 h of treatment, the CRISPR/Cas9 complex can be tracked within the nuclear of Hela cells for the purpose of gene editing of enhanced green fluorescent protein (EGFP). Moreover, TL-HFn individually packed CRISPR/Cas9 displayed higher genome editing activity compared with that of free CRISPR/Cas9 treated group both in vitro (up to 28.96%) and in vivo. Such satisfied genome editing efficiency could be attributed to the endosomal escape and pH-induced disassembly abilities given by TL-HFn after uptake into cytoplasm, which had been verified in our previous research. In all, those results prompted that TL-HFn possessed more potential for intracellular delivery of CRISPR/Cas9, with potential biocompatibility, stability and delivery efficiency.
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