Current methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such ‘MitoCRISPR’ systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.

当前针对线粒体基因组进行线粒体疾病研究和/或治疗开发的方法受到实际限制和技术灵活性的限制。人们希望有一种用于CRISPR介导的线粒体基因组编辑的分子工具箱，因为这可以利用CRISPR酶的精确性和可调节性靶向mtDNA单倍型。迄今为止描述的这种“ MitoCRISPR”系统缺乏可重复性和独立的确证。我们已经通过CRISPR核酸酶工程技术探索了人类细胞对MitoCRISPR的需求，包括使用替代的线粒体蛋白靶向序列和较小的旁系同源物，以及指导（g）RNA修饰在线粒体导入中的应用。钩端螺旋体细菌ND2006（Lb）Cas12a比Cas9变体更具针对性和耐受性。我们还提供了即使在没有靶向RNA适体的情况下，Cas9 gRNA与HeLa细胞和分离的酵母线粒体中的线粒体缔合的证据。我们的数据将线粒体靶向的LbCas12a / crRNA的mtDNA拷贝数增加，具体取决于DNA结合和切割活性。我们讨论了再现性问题以及MitoCRISPR必要的未来步骤。