LAUSR

Dear Editor, Mitochondrial diseases could be caused by heritable mutations in both mtDNA and nuclear DNA. Because mitochondria membrane insulated mtDNAs’ access from large ribonucleoprotein complex of Cas9 and sgRNA, mtDNAs are intractable for genetic modifications with RNA-guided CRISPR/Cas9 system commonly used for effectively editing nuclear DNA of different species. Although ZFN and TALEN have been previously engineered to successfully cut and eliminate mtDNA in a programmable way, correction of disease-causing point mutations in mtDNA remained challenging. Recently, DddA-derived cytosine base editors (DdCBEs) have been developed to specifically induce C-to-T conversion in mtDNA by fusion of sequence-programmable TALE and split deaminase derived from interbacterial toxins. DdCBE has been demonstrated to generate mutant zebrafish and mice carrying base-modified mtDNA by injecting DdCBE mRNA into zygotes. However, the efficacy of DdCBE for specific installation of C-to-T conversion in mtDNA of human embryos remained to be investigated. In this study, we sought to test DdCBE’s ability for base editing of mtDNA in human embryos. We first designed a pair of TALE recognizing 34 bp sequences of ND4 gene on mitochondrial genome. Split deaminase pair DddAhalf G1397-C and G1397-N, along with SOD2/COX8A mitochondrial targeting signal and uracil glycosylase inhibitor, were further fused with the TALE pair respectively to generate ND4-DdCBE that targets ND4 locus. After in vitro transcription, we injected the DdCBE mRNA into clinically discarded human embryos with three pronuclei (3PN) to check protein expression and cellular localization. Immunostaining results using antiHA and anti-FLAG antibodies revealed high expression of the DddA-TALE fusion deaminase pairs and their proper co-localization with mitotracker signal in the blastomere cell of human embryos (Supplementary Fig. S1a), similar to the recent finding in HEK293T cells. Our previous study with cytosine and adenine base editors in human embryos showed that cleaving embryos enable robust base conversion in nuclear DNA. To determine the optimal embryonic stage for mtDNA editing, we injected ND4DdCBE mRNA into human zygote, 2-cell, 4-cell and 8-cell embryos, respectively, and performed genotyping analysis 48 h post injection (Fig. 1a). We found that ND4-DdCBE exhibited detectable activity for C4 position, and injection of human embryos at the cleavage stage significantly improved base editing efficiency of ND4-DdCBE, compared with the injection at the zygote stage (Fig. 1b; Supplementary Fig. S1c). To confirm the finding with ND4-DdCBE, we further constructed DdCBE targeting ND6, ND1, ND5.1, and ATP8 genes on mitochondrial genome. By injection of ND6-, ND1-, ND5.1-, or ATP8DdCBE mRNA into human 3PN-deriverd zygote and cleaving embryos, we found DdCBE also induced efficient C-to-T base conversions and injection of DdCBE at 8-cell stage showed dramatic increase of the base conversion rate (Supplementary Figs. S1c and S2a–d). In particular, human embryos at 8-cell stage could support up to 60% cytosine conversion compared with less than 10% cytosine conversion at other stages (Supplementary Figs. S1c and S2a–d). Moreover, we showed that the advantage of using