The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

Abstract

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

The potential of mitochondrial genome engineering

“…Mutations in MT-Nd5 (ND5) generated by delivering DdCBE mRNA in mouse embryos were maintained throughout development and successfully transmitted to offspring (F1) 105 . Furthermore, one published study 106 and a pre-print 107 reported DdCBE-mediated mtDNA base editing in zebrafish embryos.…”

Mycotoxins’ Toxicological Mechanisms Involving Humans, Livestock and Their Associated Health Concerns: A Review

“…Indeed, the mycotoxins covered here include aflatoxins (aflatoxins B1, B2, G1, G2, M1, M2) [ 2 , 39 ], ochratoxin A [ 40 , 41 ], deoxynivalenol (DON) [ 42 ], fumonisins (fumonisins B1, B2, B3, B4) [ 43 , 44 , 45 , 46 ], zearalenone (ZEA), also known as F-2 mycotoxin [ 3 , 47 ], patulin, citrinin [ 38 , 42 ], ergot alkaloids [ 25 ], and T-2, which is a trichothecene mycotoxin [ 48 , 49 , 50 ]. Other mycotoxins captured in Table 2 include diacetoxyscirpenol (DAS) or 4,15-diacetoxyscirpenol (DAS), also referred to as anguidine [ 51 , 52 , 53 ], fusarenon X (FusX) [ 54 , 55 , 56 ], and nivalenol (NIV) [ 57 , 58 ]. In subsequent sections, we discuss these mycotoxins in greater detail with respect to their mechanisms of action, as well as the ailments they cause.…”

“…Fusarenon X (FusX) is a trichothecene capable of causing cytotoxicity, carcinogenicity, and immunosuppressive response in animal models and possibly in humans. It has been shown to be toxic to several types of cells, including murine thymocytes, gastric epithelial cells, and lymphocytes, along with a high toxicity to human hepatoblastoma cells [ 54 , 56 , 166 ]. In vitro and in vivo, FusX initiates apoptosis in mouse thymocytes, which may be hypothetically applicable to humans [ 53 , 55 ].…”

“…Additionally, with regard to genome science, FusX is a quick one-step transcription activator-like effector assembly system [ 167 ]. The FusX TALE Base Editor has been studied and reported for quick mitochondrial DNA programming of zebrafish disease and human cell models in vivo and in vitro, respectively, [ 56 ].…”

An optimized FusX assembly-based technique to introduce mitochondrial TC-to-TT variations in human cell lines

“…This tool is designed to be easily modified to work in diverse applications where editing of mitochondrial DNA is desired. For complete details on the use and execution of this protocol, please refer to Sabharwal et al. (2021) and Ma et al.…”