Geração de linhagens de Leishmania braziliensis nocaute (KO) para IP6K, TERT e IP6K + TERT utilizando a abordagem CRISPR/Cas9

• Autor(a) principal: Silva, Vitor Luiz da
• Data de Publicação: 2022
• Tipo de documento: Trabalho de conclusão de curso
• Idioma: por
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://hdl.handle.net/11449/217705
• topic: CRISPR/Cas9; Leishmania braziliensis; Pirofosfatos de Inositol

Leishmaniases refers to a group of diseases caused by parasites of the Leishmania genus, which mainly affects people in a social vulnerability state. Currently, the drugs available for the treatment of leishmaniasis contribute to the appearance of other clinical comorbidities due to their side effects. Therefore, the search for new therapeutic targets is extremely relevant. In model eukaryotes, inositol pyrophosphates (PP-IPs), mainly IP7 and IP8, are involved in a wide range of cellular processes, such as regulation of telomere length and homologous recombination (HR). However, the target proteins of PP-IPs, as well as their mechanism of action, are still open questions. IP7 and IP8 are synthesized by pathways involving the participation of kinases IP6K and PP-IP5K, respectively. Trypanosomatids do not have orthologous genes for PP-IP5K, i.e., they probably do not synthesize IP8, which makes them excellent models for the study of IP7. Thus, this project consists of generating lineages of Leishmania braziliensis knockout (KO) for IP6K (IP6K-/- /IP6K-/+), telomerase (TERT-/- ) and double-KO (IP6K-/- /IP6K-/+ + TERT-/- ), which will facilitate further investigation of the role of PP-IPs and their possible correlation with telomeres. These L. braziliensis lineages will be essential to answer fundamental questions, such as: how long is L. braziliensis able to survive in the absence of IP7 and telomerase? Is there any crosstalk between PP-IPs and telomeres in Leishmania? Thus, during the development of this project, we used in silico tools to elaborate small guide RNAs (sgRNAs) and specific DNA donors for the application of CRISPR/Cas9 system and consequent genome editing of L. braziliensis. After transfection and clonal selection of transfectants, we confirmed the lineages generated by PCR, and RT-PCR. Furthermore, we characterized the generated lineages through growth curves and DNA content analyzes by flow cytometry. Obtaining these lineages will pave the way for the development of other projects aimed at understanding the role of PP-IPs in DNA metabolism (especially in telomeric dynamics), which may contribute to the discovery of new therapeutic approaches aimed at parasitic elimination, since kinases are excellent targets for drug development and telomeres are considered the molecular clock of eukaryotic cells.