Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Abstract

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.

Leigh Syndrome: A Tale of Two Genomes

“…Studies using ZFN ( Minczuk et al, 2008 ; Gammage et al, 2014 , 2016 , 2018 ) and TALENS ( Bacman et al, 2013 ; Reddy et al, 2015 ; Yang et al, 2018 ) have also been used to successfully target pathogenic variants in the mitochondrial genome to eliminate these mutant genes and restore wild-type phenotype. Although a lot of these techniques have been readily adapted to editing the nuclear genome, slight modifications have to be made to target them to mitochondria to modify mtDNA ( Hussain et al, 2021 ). For instance, while CRISPR/Cas9 has been adopted for base editing in the nuclear genome, it has been challenging to do the same with the mitochondrial genome because of the difficulty associated with the delivery of guide RNA into the mitochondria ( Greenfield et al, 2017 ; Hussain et al, 2021 ; Yang et al, 2021 ).…”

“…Although a lot of these techniques have been readily adapted to editing the nuclear genome, slight modifications have to be made to target them to mitochondria to modify mtDNA ( Hussain et al, 2021 ). For instance, while CRISPR/Cas9 has been adopted for base editing in the nuclear genome, it has been challenging to do the same with the mitochondrial genome because of the difficulty associated with the delivery of guide RNA into the mitochondria ( Greenfield et al, 2017 ; Hussain et al, 2021 ; Yang et al, 2021 ). However, a CRISPR-free technology involving the use of bacterial cytidine deaminase toxin has been recently developed for use in mitochondrial base editing ( Mok et al, 2020 ).…”

Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?

“…As such, CRISPR-Cas9 technology has quickly been coupled with iPSCs to study disease-causing genetic variation in humans. Until recently, targeting of the mtDNA with CRISPR-Cas9 was unfeasible due to challenges in targeting the guide RNA and Cas9 protein to the mitochondrial matrix [74][75][76]. Consequently, most studies to-date have utilized CRISPR-Cas9 technologies to edit nDNA-encoded mitochondrial genes.…”

“…While mtDNA gene editing using CRISPR/Cas9 remains more difficult than nuclear editing, recent studies have demonstrated success doing so. In updating the method for targeting the highly inaccessible mitochondrial matrix where the mtDNA is housed, scientists have shown that by combining (1) an endogenously expressed Cas9 with a mitochondrial targeting sequence with (2) an NADH:ubiquinone oxidoreductase chain 4 targeting guide RNA, in conjunction with an RNA transport-derived stem loop element, the mtDNA can be edited [76]. In addition to advances in CRISPR/Cas9, a non-CRISPR-based approach utilizing bacterial cytidine deaminase toxin demonstrated efficiency in catalyzing C•G-to-T•A conversions within the mtDNA with high target specificity.…”

The Contextual Essentiality of Mitochondrial Genes in Cancer

“…CRISPR-Cas9 targeting of mtDNA has been challenging due to a lack of recognition sites for the Cas9 enzyme, and the poor delivery of sgRNA into mitochondria. However, recent studies have described the generation of gene-editing systems that can be used for targeting mitochondrially-encoded mitochondrial genes (MEMGs) (Mok et al, 2020;Hussain et al, 2021). It is clear from both ethidium bromide and enzymatic depletion of mtDNA in cancer cells, that MEMGs are also required for the proliferation of many cancer cell lines (Herst et al, 2004;Kukat et al, 2008).…”