CRISPR has been wildly praised and used for its simple mechanism and efficient base-pair editing of genomic DNAs which has facilitated the exploration of genetic mutation in many diseases. However, mitochondrial DNA (mtDNA) seems out of the reach of the CRISPR-Cas9 system and mutations in the mitochondrial genome could also lead server genetic disorders mostly related to energy metabolisms. The problem of editing mtDNA using CRISPR raises from a lack of RNA intake mechanism in mitochondria, unlike the nucleus, but CRISPR requires guide RNA (gRNA) to guide the Cas9 complex to the target sequence. Recently, Nature has reported a novel editing tool designed by a collaborated group of Mougous an david Liu that allows direct base-pair editing on double-strand mtDNA: DddA-derived cytosine base editor (DdCBE).
DdCBE is initially split into 2 constructs and each contains 4 components: a mitochondrial-targeting signal (MTS) sequence, a TALE protein, a split-DddA half, and a uracil glycosylase inhibitor (UGI) protein. DddA, a bacteria toxin, is a cytidine deaminase enzyme that plays the main role in this editing process which catalyzes the conversion of the nucleotide base cytosine (C) to uracil (U) directly on a double-strand DNA. In each construct, a split-DddA half is attached to the C-terminal domain of a TALE protein which binds to adjacent target mtDNA sequence and activates DddA tox by bringing two split-DddAtox half together. One split-DddA half alone in each construct is inactive and not toxic to the cell until it is combined with the other half. MTS sequences, attached to the N-terminal domain of the TALE protein, can be recognized by mitochondria which deliver the construct across the mitochondrial membrane into the matrix and are cleaved off after the entry. When DddA is activated, UGI prevents the conversion of U back to C till the next round of DNA replication.
Compared to mitochondrion-targeted nucleases TALEN and ZFN which have been used to eliminate mtDNA mutations resulting in complete degradation of mtDNA, DdCBE can reduce mutations in mtDNA at a maximum efficiency of 50% without reducing loads of mtDNA copies where low loads of mtDNA copies could be lethal. This new tool for genetic editing not only allows us to alter mitochondrial genome to gain more understandings of the role of mtDNA mutations in complex diseases such as cancer and cellular dysfunction but also gives us insight into protein designing and more base editing candidates like DdCBE and CRISPR.