A Novel Double-Stranded DNA Deaminase-Based and Transcriptional Activator-Assisted Nuclear and Mitochondrial Cytosine Base Editors with Expanded Target Compatibility and Enhanced Activity

Bacterial double-stranded cytosine deaminase DddA-derived cytosine base editor (DdCBE) guided by transcription-activator-like effector (TALE) proteins, enables mitochondrial DNA (mtDNA) editing at constrained TC sequence-context. A DddA variant DddA11, obtained from phage-assisted evolution, displays modest editing activity with HC (H =A, C, or T) sequence compatibility, while it remains relatively unattainable for GC targets. To further improve the sequence-context constraint and editing efficiency of DdCBE, we tested the targeting features of six DddAtox orthologs in CRISPR-mediated nuclear DdCBEs (crDdCBEs) and identified a novel dsDNA deaminase-composed crDdCBEs, which was originated from a Roseburia intestinalis interbacterial toxin (riDddAtox), catalyzing targeted C-to-T editing on dsDNA without sequence constraint. Mito chondrial c ytosine b ase e ditors (mitoCBE) fused by split riDddAtox and TALE arrays also exhibited substantial editing frequencies at both TC and non-TC target sites in human mitochondrial genes. Moreover, transcriptional activator (VP64, p65, or Rta) was fused to DddAtox- or riDddAtox -mediated crDdCBEs or mitoCBEs, and these engineered crDdCBEs and mitoCBEs substantially improved both nuclear and mitochondrial DNA (mtDNA) editing efficiencies by up to 5.1- and 1.7- fold respectively on average at both HC and GC targets. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in human and mouse cells as well as in mouse embryos with conversion frequencies of up to 58% at non-TC target sites.Funding Information: This work was supported in part by National Key Research and Development Program (2021YFA0804702, X.H.; 2018YFC1004700, Y.Q.), Excellent Youth Foundation of Guangdong Scientific Committee, 2020B1515020018, Y.Q.), the leading talents of Guangdong province program (2016LJ06S386, X.H.) and Emergency Key Program of Guangzhou Laboratory (EKPG21-18, X.H.).Declaration of Interests: The authors declare no competing interests.