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What now for human genome editing?
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Applications of CRISPR-Cas9 in Alzheimer's Disease and Related Disorders.
Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease represent some of the most prevalent neurodegenerative disorders afflicting millions of people worldwide. Unfortunately, there is a lack of efficacious treatments to cure or stop the progression of these disorders. While the causes of such a lack of therapies can be attributed to various reasons, the disappointing results of recent clinical trials suggest the need for novel and innovative approaches. Since its discovery, there has been a growing excitement around the potential for CRISPR-Cas9 mediated gene editing to identify novel mechanistic insights into disease pathogenesis and to mediate accurate gene therapy. To this end, the literature is rich with experiments aimed at generating novel models of these disorders and offering proof-of-concept studies in preclinical animal models validating the great potential and versatility of this gene-editing system. In this review, we provide an overview of how the CRISPR-Cas9 systems have been used in these neurodegenerative disorders.
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Crispr/Cas9 homologous recombination (HR).
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Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9.
Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.
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Therapeutic Genome Editing With CRISPR/Cas9 in a Humanized Mouse Model Ameliorates α1-antitrypsin Deficiency Phenotype.
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
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CRISPR/Cas9, the Powerful New Genome-Editing Tool for Putative Therapeutics in Obesity.
The molecular technology known as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is revolutionizing the field of medical research and deepening our understanding of numerous biological processes. The attraction of CRISPR/Cas9 lies in its ability to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms, a technology that was once considered either too expensive or scientifically risky. CRISPR/Cas9 has been successfully applied in agriculture to develop the next generation of disease-resistant plants. Now, the capability of gene editing has been translated to the biomedical area, focusing on the future of medicine faced with drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, for example, or finding the ultimate strategy for cancer or HIV. In this regard, it was recently demonstrated that an injection of cancer-fighting CRISPR-modified white blood cells in a patient suffering from metastatic lung cancer could lead to promising results. Researchers and bioethicists are debating questions about the regulation of CRISPR/Cas9 that must be addressed. While legal challenges surround the use of this technique for genetically modifying cell lines in humans, we review the basic understanding of CRISPR/Cas9 and discuss how this technology could represent a candidate for treatment of non-communicable diseases in nutrition, such as obesity.
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CRISPR-Cas9 Gene-Edited Therapy Shows Sustained Treatment Response.
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Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.
Metabolic engineering frequently requires both gene knockouts and gene integration. CRISPR-Cas9 has been extensively used to create double-stranded DNA breaks that result in indel mutations; however, such mutations can revert or create toxic product. Gene integration can also be accomplished by CRISPR-Cas9 introduced double-stranded DNA breaks and a donor DNA cassette. Here we describe our protocol for combining an efficient gene knockout created by introducing DNA cuts with two guide RNAs with a gene to be integrated at the knockout site. Including guide RNA target sites flanking the homology regions around the gene to be integrated enables both homology-directed repair and homology-mediated end joining, resulting in few deletions and a significant proportion of correctly knocked out and integrated genes.
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The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.
Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.
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An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing.
Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/ybx1) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.
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