Reducing off-target effects of DdCBEs by reversing amino acid charge near DNA interaction sites
Long Xie, Yaqi Cao, Di Li, Mengxue Ma, Danrong Jiao, Feng Hu, Zhenrui Zuo, Erwei Zuo
Abstract
DddA tox -derived cytosine base editors (DdCBEs) can mediate precise, CRISPR-independent editing of mitochondrial DNA (mtDNA), but were recently shown to potentially introduce extensive and non-trivial off-target effects throughout the genome, 1 , 2 presenting a significant obstacle for research applications and an obvious safety issue for use in clinical therapies of hereditary mitochondrial disorders. Here, utilizing the crystal structure of DddA tox in complex with DNA, we predicted DNA-binding sites on the DddA tox protein surface and generated variants by converting positively charged residues at these sites to negatively charged amino acids. By modifying the DNA-binding capability of DddA tox through charge reversal, especially through K1402D or K1402E conversions, we could generate split-architecture DdCBEs with high on-target editing efficiency at multiple sites in mtDNA and significantly fewer off-target effects in both mtDNA and whole genome of mice, ~400 times lower than wild-type (WT) DdCBE (DdCBE WT ). This study establishes K1402D/K1402E-DdCBE (DdCBE K1402D/E ) editors as efficient, high-fidelity, and relatively safe research tools for mitochondrial diseases that warrant exploration for therapeutic applications, and demonstrates a strategy for reducing off-target effects in DddA tox family base editors.