Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

• Main Authors: Syed-Rehan A. Hussain, Mehmet E. Yalvac, Benedict Khoo, Sigrid Eckardt, K. John McLaughlin
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2021-04-01
• Series: Frontiers in Genetics
• Subjects: mitochondria; heteroplasmic mutations; PNPase; RP-loop; chimeric guide RNA
• Online Access: https://www.frontiersin.org/articles/10.3389/fgene.2021.627050/full

Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.