Mechanisms of replication and repair in mitochondrial DNA deletion formation

Deletions in mitochondrial DNA (mtDNA) are associated with diverse human pathologies including cancer, aging and mitochondrial disorders. Large-scale deletions span kilobases in length and the loss of these associated genes contributes to crippled oxidative phosphorylation and overall decline in mitochondrial fitness. There is not a united view for how mtDNA deletions are generated and the molecular mechanisms underlying this process are poorly understood. This review discusses the role of replication and repair in mtDNA deletion formation as well as nucleic acid motifs such as repeats, secondary structures, and DNA damage associated with deletion formation in the mitochondrial genome. We propose that while erroneous replication and repair can separately contribute to deletion formation, crosstalk between these pathways is also involved in generating deletions.

P09.06 ‘An enhanced CRISPR tool for treating chronic myelogenous leukemia’

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplastic disease, occurring in 1 to 2 cases per 100.000 adults, which accounts this type of cancer for approximately 15% of newly diagnosed leukemia in adult patients. The diagnosis is based upon the genetic translocation between the t(9;22)(q34;q11.2), resulting in formation of Philadelphia fusion chromosome, coding for BCR-ABL1 oncoprotein. The life-long treatment relies on using tyrosine kinase inhibitors (TKIs). In some cases, patients develop point mutations, leading to resistance to TKIs treatment, nearly in 2%. Allogeneic stem cell transplantation is the possible solution for these individuals in late stages of CML with success cure rate only approximately at 40%.1 Based on this funding new solutions for treating cancer with genetic etiology are considered. CRISPR/Cas system, composed of guide RNA, targeting endonuclease Cas9 to specific target genomic region has been used before to mediat breakage of Philadelphia chromosome at the site of oncogenic translocation, although at lower efficiency.2Materials and MethodsK562 cells, model for Philadelphia chromosome positive cells, were used. Constructs, expressing BCR-ABL1 targeting gRNA and Cas9, tethered via coiled-coil forming peptides to E.coli exonuclease EXOIII, were nucleofected into target cells. T7E1 assay to detect genome modifications was carried out. TUNEL assay, FACS analysis and bioluminescence measurement were used for cell death determination. SCID mice were used for a subcutaneous K562 cancer model.ResultsOur strategy was to couple Cas9 to the exonuclease to promote large deletion at the target site. Of the different exonucleases tested, the EXOIII exhibited the best performance in terms of deletion formation. To improve the rate of deletion genetic lesions, we connected Cas9 and EXOIII via coiled-coil forming peptides, bringing the two enzymes into close proximity (CRISPR-EXO). This resulted in an increased deletion formation compared to the standard CRISPR/Cas system. We performed a case study for the use of the CRISPR-EXO system as a potential anti-cancer therapeutic tool. In the case of our new system, we showed significant increase in cell death due to higher genome modification in BCR-ABL1 region. Later, these findings were confirmed also in an animal cancer model, where animals with tumors, electroporated with CRISPR-EXO system showed full survival and drastic reduction in tumor size.ConclusionsCRISPR-EXO upgraded CRISPR system based on tethering Cas9 protein to exonuclease EXOIII by heterodimeric coiled-coil forming peptides, resulted in highly efficient editing of BCR-ABL1 fusion gene, leading to enhanced death of CML cancer cells.ReferencesJabbour E, Kantarjian H. Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring. Am J Hematol2018; 93: 442–459.Lekometsev S, Aligianni S, Lapao A, Bürckstümmer T. Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology. BMC Genomics 2016; 17: 739–745.Disclosure InformationD. Lainšček: None. V. Forstnerič: None. Š. Malenšek: None. M. Skrbinek: None. M. Sever: None. R. Jerala: None.

Risk of mitochondrial deletions is affected by the global secondary structure of the mitochondrial genome

Ageing is associated with accumulation of somatic mutations. This process is especially pronounced in mitochondrial genomes (mtDNA) of postmitotic cells, where the accumulation of somatic mitochondrial deletions is associated with healthy ageing and mitochondrial encephalomyopathies. Deletions are often flanked by direct nucleotide repeats, however, they do not provide an exhaustive explanation of deletion distribution. We hypothesized that in parallel with the role of direct repeats there is also a global secondary structure of mtDNA, shaping deletion formation. Analyzing the folding energies of the heavy chain, which stays single-stranded during mtDNA replication, we observed a potential contact zone between 6-9kb and 13-16kb of the major arc of mtDNA. Describing the distribution of deletions in the human mtDNA we demonstrated that the contact zone is 3-times more mutagenic under all else equal. The proposed topological model improves our understanding of the mechanisms of deletion formation in the human mitochondrial genome.