HACKING THE HUMAN MITOCHONDRIAL GENETIC CODE

Original title

ТЕХНОЛОГИИ ВЗЛОМА ГЕНЕТИЧЕСКОГО КОДА МИТОХОНДРИЙ ЧЕЛОВЕКА

Authors

I.О. Мazunin

Contact information

IK BFU, Kaliningrad, Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.

Pages

63-64

DOI

10.31255/978-5-94797-318-1-63-64

Abstract

Despite significant progress in the development of nuclear DNA editing technologies, the latter have not been made applicable to mitochondrial DNA (mtDNA) yet. However the creation of mitochondrial editing systems would solve many existing fundamental and applied dilemas. The only hitherto described approaches of human mtDNA mutation elimination in cell lines use site-specific nucleases. Recent ones include mitochondria-targeted restriction endonucleases (mitoREs), mitochondria-targeted Zinc-fingers (mitoZFNs), and mitochondria-targeted TALENs (mitoTALENs). All these approaches are based on specific protein-nucleic acid interactions to cut out target mtDNA. RNA-guided endonuclease (RGEN) technologies seem to be more flexible and offer perspective due to their reliance on Watson-Crick interactions for specific mtDNA site recognition. The limitation on the ubiquitous use of RGEN technologies is their two-component nature: the systems include protein and RNA-parts. Successful delivery of RNA moieties into mitochondria is very complicated but there is experimental proof. Moreover homology-directed repair relies on donor DNA as a homologous repair template.

We have shown that when modified with a mitochondrial target signal SpCas9 (MitoCas9) and AsCpf1 (MitoAsCpf1) can be effectively imported into the mitochondrion of human cells. We verified its mitochondrial localization by immunocytochemistry and western blotting. Also we built several plasmid vectors that contain one of the RNA import determinants (there are four determinants for RNA import into human mitochondria called MRP, RP, HD and HF) added to guide RNA (gRNA). We have shown that the addition does not affect the gRNA-AsCpf1 and gRNA-SpCas9 interaction and the whole complex’s cleavage activity in vitro. To verify mitochondrial localization of the modified gRNAs we used one-step RT-ddPCR method and northern blotting. To estimate the mtDNA copies in cells treated by gRNAs-MitoCas9 or gRNA-MitoAsCpf1 we performed ddPCR analysis, adapted for that. Phoenix cells treated with gRNA-HF and MitoCas9 had a significant decrease in mtDNA copies (p < 0.01). Next we are going to estimate the mtDNA copies in cells treated with one of the modified gRNAs and MitoAsCpf1.

To increase RNA import efficiency we have decided to encode the RNA moieties into a DNA structure and import it into mitochondrion using the TOM/TIM protein pathway. We would like to present the system called mitoTALE which, as we think, could deliver dsDNA molecules into the mitochondrion. The system includes two parts: the modified DNA-binding protein (recombinant protein which consists of a TAL effector fused with a mitochondrial target signal in its N-tail) and a binding site at the 5'-end of the imported dsDNA molecule. Imported dsDNA are supposed to be expressed inside of the mitochondrion as functional genetic code, and could be used as a DNA template for homology-directed recombination or/and additional genetic material. So here we are presenting and discussing our research on mitochondrially modified RGEN technologies in the context of mtDNA level manipulation as a potential future strategy for therapeutic intervention in selected mitochondrial diseases. This work was supported by the Russian Science Foundation [grant numbers 17-75-20015].