LAUSR

Dear Editor, Mitochondrial DNA (mtDNA) is encapsulated by the organelle membrane forming the barrier for the access of CRISPR-based gene-editing tools to the mtDNA. Furthermore, mitochondrial genome lacks similar repair systems for the protection of nuclear genome from DNA damage after induction of double-strand break by programmable nuclease such as ZFN, TALEN etc., which results in the elimination of target mtDNA instead of mutation installation on mtDNA in contrast to the outcome of indel formation on nuclear DNA. Recent studies positioned DdCBE as a promising technology to install targeted mutations or introduce transmissible mutations of base conversion in mammalian mtDNA rather than eliminate them with previous ZFor TALE-based nuclease. Thus, DdCBE has the potential to model mitochondrial disease mutations, correct pathogenic variants, and expand our knowledge of mitochondrial biology. However, it is worth mentioning that these studies have found that DdCBE can cause low-frequent off-target events on mtDNA. As indicated, the off-target profile of DdCBE remained to be comprehensively investigated by additional research for their systematic effect on mtDNA as well as nuclear genome. In the current study, we performed the GOTI (genomewide off-target analysis by two-cell embryo injection) method previously developed by us to evaluate the off-target effect of DdCBE on both mtDNA and nuclear DNA modification. At first, we in vitro transcribed two pairs of DdCBE mRNA targeting the mtDNA ND5 gene (G12918 and C12336) and injected them with Cre mRNA into one blastomere of two-cell embryos derived from Ai9 background leaving another blastomere uninjected (Fig. 1a and Supplementary Fig. S1a). Thereby, Cre-activated tdTomato fluorescence will distinguish DdCBE-injected cells from non-fluorescent uninjected cells derived from the same two-cell Ai9 embryos. At 14.5 days after transferring injected 2-cell embryos into surrogate female mice, we collected E14.5 embryos to sort tdTomato and tdTomato cells for genotyping base conversion outcomes on two targeted loci of mtDNA as well as whole-genome sequencing (WGS) analysis (Fig. 1a). Sanger sequencing and Targeted deep sequencing results showed efficient mtDNA editing by DdCBE with m.G12918A and m. C12336T conversion rate of up to 46% in both non-sorted and sorted tdTomato tissues, contrasting with the only wild-type alleles detected in tdTomato tissues (Supplementary Fig. S1b, c and Table S1). For G12918 and C12336-targeting DdCBE, we also observed higher editing rate of up to 72% for sorted cells than unsorted ones on the basis of WGS (Fig. 1b, c). Furthermore, there are several unintended and sequence-independent C-to-T editing events identified by WGS analysis with lower than 5% frequency centered around m.G12918A or m. C12336T on mtDNA (Supplementary Fig. S2a). For all unintended editing events, some fall within spacer