Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 37857619.
- Also identified by DOI 10.1038/s41467-023-42359-3 and PMC identifier 10587121.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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 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 DddI<sub>A</sub>, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddI<sub>A</sub>. 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.
Medical subject headings
- Gene Editing
- Mitochondria