Targeted deletions in human mitochondrial DNA engineered by Type V CRISPR-Cas12a system

Mutations in mitochondrial DNA (mtDNA) contribute to various neuromuscular diseases, with severity depending on heteroplasmy level when mutant and wild-type mtDNA coexist within the same cell. Developing methods to model mtDNA dysfunction is crucial for experimental therapies. Here, we adapted the Type V CRISPR-AsCas12a system, which recognizes AT-rich PAM sequences, for targeted editing of human mtDNA. We show that AsCas12a effector, fused with a mitochondrial targeting sequence (MTS) from Neurospora crassa ATPase subunit 9, is efficiently addressed into human mitochondria and induces specific mtDNA cleavage in human cells. As a proof-of-concept, we demonstrate that AsCas12a, complexed with two crRNAs targeting distant regions of human mtDNA, introduces specific deletions in mtDNA. For the first time, we provide experimental data proving that a CRISPR system can be used not only for mtDNA degradation but also for precise mtDNA manipulation, offering a potential therapeutic avenue to address mitochondrial disorders.