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Developing methods to model mtDNA dysfunction is crucial for experimental therapies. Here, we adapted the Type V CRISPR\u2013AsCas12a system, which recognizes AT-rich PAM sequences, for targeted editing of human mtDNA. We demonstrated that mitochondrial targeting sequence (MTS) from Neurospora crassa ATPase subunit 9 efficiently addressed the AsCas12a effector nuclease into human mitochondria. When programmed with two CRISPR RNAs (crRNAs) targeting distant regions of mtDNA, the mito-AsCas12a can cleave mtDNA, enabling generation of deletions in cultured human cells. Next generation sequencing of the deletions boundaries confirmed mtDNA ligation after the cleavage by the mitoCRISPR\u2013AsCas12a system. Therefore, we provide experimental data proving that a CRISPR system has potential to be used for precise mtDNA manipulation, offering a promising tool for generating predefined deletions in mtDNA and creating cellular models of mitochondrial disorders.<\/jats:p>","DOI":"10.1093\/narmme\/ugaf021","type":"journal-article","created":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T07:33:32Z","timestamp":1748936012000},"source":"Crossref","is-referenced-by-count":9,"title":["Targeted deletions in human mitochondrial DNA engineered by Type V CRISPR\u2013Cas12a system"],"prefix":"10.1093","volume":"2","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4893-7644","authenticated-orcid":false,"given":"Natalia","family":"Nikitchina","sequence":"first","affiliation":[{"name":"UMR7156\u2014Molecular Genetics, Genomics, Microbiology, CNRS\/University of Strasbourg , Strasbourg\u00a0 67000 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne-Marie","family":"Heckel","sequence":"additional","affiliation":[{"name":"UMR7156\u2014Molecular Genetics, Genomics, Microbiology, CNRS\/University of Strasbourg , Strasbourg\u00a0 67000 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikita","family":"Shebanov","sequence":"additional","affiliation":[{"name":"UMR7156\u2014Molecular Genetics, Genomics, Microbiology, CNRS\/University of Strasbourg , Strasbourg\u00a0 67000 ,","place":["France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilya","family":"Mazunin","sequence":"additional","affiliation":[{"name":"Petrovsky Medical University Department of Biology and Genetics, , Moscow\u00a0 119435 ,","place":["Russia"]},{"name":"Research Centre for Medical Genetics , Moscow\u00a0 115478 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