Mitochondrial diseases are a major genetic disease. The prevalence rate among newborns in the United States is about 1 in 5000. China currently lacks complete epidemiological data on mitochondrial diseases. At present, nearly 300 genes that cause mitochondrial diseases are known, including both mitochondrial DNA (mtDNA) coding and nuclear DNA (nDNA) coding. Although mtDNA only encodes 37 genes, due to the lack of nucleosomal protection, since mtDNA mutations were first reported in 1988 to cause maternally inherited mitochondrial diseases, more than 270 mitochondrial DNA mutations have been found to be related to mitochondrial diseases.
Mitochondrial diseases involve multiple tissues such as the nervous system, skeletal muscle, and myocardium. The patients usually get onset in infants and young children. Most children will die due to the failure of the respiratory system and heart and other important organs before they reach adulthood. They are a type of serious harm to human health. Of disabling and fatal diseases. Due to the large number of pathogenic genes in mitochondrial diseases, the various pathogenic mechanisms have brought great challenges to the research and development of clinical treatment strategies. There are no effective therapeutic drugs and treatment methods available. The development of gene editing technology has brought dawn to the research and treatment of mitochondrial diseases. For mitochondrial diseases caused by nDNA mutations, the base editors and lead editors developed based on the CRISPR system provide powerful tools for disease model construction and gene therapy; but for mitochondrial diseases caused by mtDNA mutations, due to the Guide RNA cannot enter the mitochondria, and the above tools are helpless for mtDNA mutation models and gene therapy. In July 2020, David R. Liu’s laboratory developed the base editor DdCBE for mtDNA editing by combining the core region of double-stranded DNA deaminase derived from Burkholderia spp. DddAtox and TALE, and used it in cell lines. The editing from C∙G to T∙A is realized, which provides the possibility for the construction of animal models and gene therapy of mitochondrial diseases caused by mtDNA mutations.
Recently, Shen Bin’s laboratory from Nanjing Medical University-State Key Laboratory of Reproductive Medicine and Lou Xin’s research group from Nanjing University School of Medicine-Institute of Model Animals jointly published an online publication titled “ Precision modeling of mitochondrial diseases in zebrafish via” on Cell Discovery . DdCBE-mediated mtDNA base editing article. The research constructed a DdCBE high-efficiency assembly and screening platform, realized for the first time heritable mtDNA high-efficiency gene editing on zebrafish, and accurately simulated human mtDNA pathogenic mutations, providing a good foundation for the pathogenic mechanism and gene therapy of mitochondrial diseases .
The traditional TALE assembly method is time-consuming and laborious. In order to quickly assemble the TALE-based DdCBE vector, the author constructed 192 RVD libraries, 4 mitochondrial-localized backbone expression vectors and 4 nuclear-localized backbone expression vectors, using the Golden Gate method The DdCBE carrier is assembled in one step.
In order to mimic the human mtDNA pathogenic mutations G8363A, G3733A and G13513A, through homology, the NLS-DdCBE vector targeting zebrafish mtDNA G8892, G4247 and G14076 sites and the T vector containing the target sequence were designed and screened on HEK293FT cells. Create an efficient DdCBE combination. By injecting high-efficiency MTS-DdCBE combination mRNA into zebrafish one-cell stage fertilized eggs, individuals with target mutations were successfully constructed. The mutation rate can be as high as 88.32%, and these mutations can be passed on to offspring through female fish. The zebrafish carrying the G4247A and G14076A mutations have significantly reduced locomotor ability. Transmission electron microscopy can be used to observe severe damage to the mitochondrial ridges of the mutant zebrafish. Through sequencing of whole mitochondrial DNA, it is found that DdCBE induces a small amount of off-target, and these off-target sites are enriched near the target site, which may be caused by the instability of TALE binding.
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