Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library
Haifeng Sun, Zhaojun Wang, Limini Shen, Yeling Feng, Han Lu, Xuezhen Qian, Runde Meng, Kangming Ji, Dong Liang, Fei Zhou, Xin Lou, Jun Zhang, Bin Shen
Abstract
Abstract DddA-derived cytosine base editors (DdCBEs) greatly facilitated the basic and therapeutic research of mitochondrial DNA mutation diseases. Here we devise a saturated spacer library and successfully identify seven DddA homologs by performing high-throughput sequencing based screen. DddAs of Streptomyces sp. BK438 and Lachnospiraceae bacterium sunii NSJ-8 display high deaminase activity with a strong G C context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to A C context. We also find that different split sites result in wide divergence on off-target activity and context preference of DdCBEs derived from these DddA homologs. Additionally, we demonstrate the orthogonality between DddA and DddI A , and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddI A . The current study presents a comprehensive and unbiased strategy for screening and characterizing dsDNA cytidine deaminases, and expands the toolbox for mtDNA editing, providing additional insights for optimizing dsDNA base editors.