Engineering targeted deletions in the mitochondrial genome

Abstract

ParagraphMitochondria are a network of critical intracellular organelles with diverse functions ranging from energy production to cell signaling. The mitochondrial genome (mtDNA) consists of 37 genes that support oxidative phosphorylation and are prone to dysfunction that can lead to currently untreatable diseases. Further characterization of mtDNA gene function and creation of more accurate models of human disease will require the ability to engineer precise genomic sequence modifications. To date, mtDNA has been inaccessible to direct modification using traditional genome engineering tools due to unique DNA repair contexts in mitochondria 1 . Here, we report a new DNA modification process using sequence-specific transcription activator-like effector (TALE) proteins to manipulate mtDNA in vivo and in vitro for reverse genetics applications. First, we show mtDNA deletions can be induced in Danio rerio (zebrafish) using site-directed mitoTALEnickases (mito-nickases). Using this approach, the protein-encoding mtDNA gene nd4 was deleted in injected zebrafish embryos. Furthermore, this DNA engineering system recreated a large deletion spanning from nd5 to atp8, which is commonly found in human diseases like Kearns-Sayre syndrome (KSS) and Pearson syndrome. Enrichment of mtDNA-deleted genomes was achieved using targeted mitoTALE-nucleases (mitoTALENs) by co-delivering both mito-nickases and mitoTALENs into zebrafish embryos. This combined approach yielded deletions in over 90% of injected animals, which were maintained through adulthood in various tissues. Subsequently, we confirmed that large, targeted deletions could be induced with this approach in human cells. In addition, we show that, when provided with a single nick on the mtDNA light strand, the binding of a terminal TALE protein alone at the intended recombination site is sufficient for deletion induction. This "block and nick" approach yielded engineered mitochondrial molecules with single nucleotide precision using two different targeted deletion sites. This precise seeding method to engineer mtDNA variants is a critical step for the exploration of mtDNA function and for creating new cellular and animal models of mitochondrial disease.

The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

“…None of these mutations were embryonically lethal, allowing us to monitor the germline transmission of these edits in subsequent F1 generations during the future course of the study. It will be interesting to see if such high percentage of edits are able to withstand the oocyte selection of mutant mtDNA as observed previously with low heteroplasmy of mtDNA large-scale deletion in zebrafish 16 . Recently, a study describing mice editing with a custom designed DdCBE demonstrated base edits in mice F0 and F1 embryos albeit with maximum mtDNA editing efficiency up to 57% in F0 generation for one of the loci.…”

“…To quantify the editing efficiency in vertebrate zebrafish embryos, a cassette containing DddA tox was cloned at the 3’ end of the C-terminus of the TALE domain in the linearized pk- idh2 GoldyTAL-lacZ-Fok1 destination vector. We identified a highly functional and sufficient mitochondrial targeting sequence from a reverse engineered in vivo protein trap in a nuclearly encoded mitochondrial protein 16,24 . This derived minimal mitochondrial targeting sequence (MTS) was highly active in zebrafish in vivo compared to previously described MTS sequences and was used to target an array of different proteins capable of interacting with the mtDNA genome 16 .…”

“…The FusX system can be automated and integrated to a high throughput robotic liquid handling device, thereby scaling up the number of TALE base editors that can be assembled with minimal human error. This system is available at Addgene and has been successfully utilized for nuclear and mitochondrial gene editing applications, both in vitro and in vivo , with high efficiency 16, 19 .…”

“…Mitochondrially targeted nucleases selectively bind the mtDNA harboring deletions/edits and introduce double stranded breaks, thereby triggering rapid degradation of mtDNA. In a previous study by our group, we developed an alternative approach to introduce precise deletions in the mtDNA using a novel “block and nick” approach to model KSS in zebrafish 16 . However, the utility of these nuclease-based gene editing systems cannot be extended to correct or model homoplasmic and single nucleotide pathogenic mtDNA variations.…”

Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates

“…Also, endonucleases imported into mitochondria can shift heteroplasmy ratios to alter mitochondria and cell functions by targeting specific sequences for destruction. However, these endonucleases are laborious to produce, are limited to certain mtDNA sequences, are inefficient, and do not yield homoplasmy ( Campbell et al, 2018 ; Yahata et al, 2017 ; Yang et al, 2018 ). Of note, a recent and exciting development using a bacterial cytidine deaminase, DddA, to edit mtDNA single-base sequences is tempered by low efficiency and an undesirable off-target rate ( Mok et al, 2020 ).…”