Abstract
Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.
Highlights
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology derived from bacterial adaptive immune response system, was adapted for mammalian genome editing almost a decade ago [1]
One of the most frequently used CRISPR/Cas9 applications is to generate (i) gene knock-outs (CRISPR-KO) to permanently disrupt gene expression of the targeted gene [1] and (ii) knock-ins (CRISPR-KI), where the double strand breaks (DSB) can be utilised for specific modifications by the homology-directed repair (HDR) mechanism; this requires the introduction of a donor template alongside the CRISPR/Cas9 system to facilitate the knock-in of the specific change at the targeted genomic site [6]
Large-scale CRISPR library screening allows the identification of potential therapeutic targets in the search of a cure for mitochondrial disease
Summary
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology derived from bacterial adaptive immune response system, was adapted for mammalian genome editing almost a decade ago [1] It is composed of two main components, a Cas endonuclease that mediates DNA cleavage and a single guide RNA (sgRNA) composed of a CRISPR RNA (crRNA) that defines the genomic target and a trans-activating. CRISPR screening libraries have been used to knock-out (loss-of-function CRISPR-KO), activate (gain-of-function CRISPRa), or inhibit (transcriptional repression CRISPRi) target genes [10,19,20,21,22] These approaches allow extensive interrogation of genetic factors involved in various biological pathways and mapping interacting networks between the genes of interest to delineate unknown molecular mechanisms associated with specific cellular phenotypes. Genome-wide pooled CRISPR screens have been mainly performed in cancer cell lines, but the application of CRISPR libraries to human-derived-induced pluripotent stem cells (iPSCs) has an immense potential to reveal molecular mechanisms and/or drug targets in various diseases including mitochondrial disease
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