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CRISPR-based gene therapy for huntington's disease: current advances and future prospects.
CRISPR-Cas9 technology offers transformative potential in treating Huntington's Disease (HD) by directly addressing its genetic root causes. This manuscript explores the pathophysiological mechanisms of HD, characterized by toxic mutant huntingtin (mHTT) protein resulting from expanded CAG repeats in the HTT gene, and the challenges posed by current therapeutic limitations. We comprehensively review the mechanisms of CRISPR-based therapeutic strategies, including excision of expanded repeats, allele-specific targeting, and epigenome editing, highlighting their efficacy in preclinical studies using animal models and human iPSCs. Delivery methods, such as viral and non-viral vectors, are analysed for their role in optimizing therapeutic outcomes while minimizing off-target effects and immune responses. Ethical and safety considerations, especially regarding precision and long-term impacts, are critically examined alongside emerging strategies to enhance specificity. With ongoing clinical trials and advancements in delivery systems, CRISPR technology represents a paradigm shift in addressing HD and broader neurodegenerative conditions. This review underscores the promise of gene editing in overcoming existing barriers and paving the way for transformative therapeutic approaches.
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Genome Editing of C. elegans.
Caenorhabditis elegans, a 1 mm long free-living nematode, is a traditional model animal for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle, well-annotated genome, simple morphology, and transparency. Recently, genome editing technologies have been increasingly used in C. elegans, thereby facilitating their genetic analyses. Here, I introduce a protocol frequently used in C. elegans genome editing.
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Simultaneous precise editing of multiple genes in human cells.
When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.
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Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.
First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.
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Application of compact CRISPR/Cas nucleases for citrus genome editing.
Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.
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CRISPR/Cas Technologies Applied to Pseudogenes.
Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and by the fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible: To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.
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Commentary on "CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice".
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CRISPR-Cas systems ushered in an era of facile DNA-free genome editing.
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Revolutionizing viral resistance strategies in rice: Evolution from RNAi to precision genome editing.
Rice viruses are a major threat to global food security, causing significant yield losses in key rice growing regions. RNA interference (RNAi) has been crucial in engineering viral resistance in rice by silencing essential viral genes. However, the advent of genome editing, especially CRISPR/Cas, has transformed this field by allowing precise alterations of viral susceptibility genes, offering more durable and targeted resistance. This review examines the advances in RNAi strategies and the shift toward CRISPR/Cas technologies, highlighting how genome editing addresses RNAi's limitations, such as broader viral strain coverage and stronger resistance. These tools, integrated with advanced breeding methods, promise to develop rice varieties with durable, broad-spectrum virus resistance, contributing to sustainable rice production and food security.
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A new breakthrough in genome editing: the story of Casgevy.
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