RESUMEN
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 GC context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to AC 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 DddIA, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddIA. 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.
Asunto(s)
Edición Génica , Mitocondrias , Mitocondrias/metabolismo , Mutación , ADN Mitocondrial/genética , Citidina Desaminasa/genética , Sistemas CRISPR-Cas , CitosinaRESUMEN
Double-stranded DNA-specific cytidine deaminase (DddA) base editors hold great promise for applications in bio-medical research, medicine, and biotechnology. Strict sequence preference on spacing region presents a challenge for DddA editors to reach their full potential. To overcome this sequence-context constraint, we analyzed a protein dataset and identified a novel DddAtox homolog from Ruminococcus sp. AF17-6 (RsDddA). We engineered RsDddA for mitochondrial base editing in a mammalian cell line and demonstrated RsDddA-derived cytosine base editors (RsDdCBE) offered a broadened NC sequence compatibility and exhibited robust editing efficiency. Moreover, our results suggest the average frequencies of mitochondrial genome-wide off-target editing arising from RsDdCBE are comparable to canonical DdCBE and its variants.