A conditional knockout rat resource of mitochondrial protein-coding genes via a DdCBE-induced premature stop codon

Abstract

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system . Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.

Mitochondrial genome editing: strategies, challenges, and applications

“…In the cultured cell lines, utilizing various delivery strategies as previously mentioned, mtDNA base editors can target virtually all genes within the mtDNA, encompassing human, mouse, and rat cells. Furthermore, in vivo mtDNA base editing has been confirmed in various organisms, including mouse ( 46 , 48 , 50 , 55 , 61 , 62 , 70 ), zebrafish ( 63 , 64 ), rat ( 65 - 67 ), and even plant ( 72 ). However, the effectiveness of target achievement and efficiency may vary depending on the design rules of individual mtDNA base editors.…”

“…Electroporation is commonly used for transfecting various cell types, including human, mouse, rat, and patient-derived cells. In addition, the delivery of genome engineering tools in the form of mRNA into embryonic cells is a widely practiced method ( 24 , 46 , 48 , 55 , 61 - 67 ). This allows for mitochondrial genome editing in the early stages of embryonic development, and the generation of new models for genetic disease.…”

“…This allows for mitochondrial genome editing in the early stages of embryonic development, and the generation of new models for genetic disease. Through microinjection of embryos, mtDNA-associated disease models have been created using mitochondrial genome editing in mouse ( 46 , 48 , 55 , 61 , 62 ), zebrafish ( 63 , 64 ), and rat ( 65 - 67 ). Moreover, the efficiency and accuracy of mitochondrial genome editing have been evaluated by applying the microinjection method to clinically discarded human embryos with three pronuclei (3PN) or two pronuclei (2PN) ( 56 , 68 ).…”

Clinical Approaches for Mitochondrial Diseases

“…The mitochondrial base editor is commonly used to induce pathogenic mutations in rodents to establish a mouse model of mitochondrial disease with disease phenotypes [234,[236][237][238]. The use of DdCBEs in an in vivo model is usually conducted through microinjection at the zygote stage but cannot effectively target all mitochondria, resulting in a heteroplasmic distribution of mutated mtDNA.…”