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Endogenous tagging of Leishmania major META1 and META2 genes using CRISPR-CAS9, as a way to identify metacyclics in axenic culture

Grant number: 20/08162-3
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): October 01, 2020
Effective date (End): September 30, 2022
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal researcher:Maria Isabel Nogueira Cano
Grantee:Veronica Silva Fontes
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Associated research grant:18/04375-2 - Studies about the biogenesis and composition of the Leishmania spp. ribonucleoprotein complex and its regulation, AP.TEM

Abstract

Leishmaniases are neglected diseases caused by parasites of the genus Leishmania, for which there is no suitable treatment or control. Available therapies are expensive, presenting high toxicity levels, and can induce drug resistance in the parasites. All of these issues highlight the need to seek new therapeutic targets and to understand the parasite life cycle and the biological issues involved in each parasites' life stage, such as the metacyclogenesis. The process of metacyclogenesis occurs inside the mammalian host, and it involves both morphological and functional differentiation and it is when non-infective procyclic promastigote transforms into the highly infective metacyclic promastigote. During metacyclogenesis, parasites show differential gene expression mainly of virulence factors that are necessary for the parasite transformation. Among these genes are META1 and META2 which are expressed in all Leishmania species analyzed so far. In this project, we intend to promote the endogenous tagging of L. major META1 and META2 using CRISPR-Cas9 to facilitate the identification of metacyclic in axenic cultures. Different assays will be used to assess whether the tagged proteins will only be expressed in metacyclic forms and if the fluorescent labels alter the metacyclogenesis and parasite growth profile. In this way, this work can generate parasite cell lines that will become important tools for the research that we are conducting about the telomeric machinery during the parasite developmental cycle and may help to identify biological and molecular features that are exclusive to parasites in the metacyclic form.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
ASSIS, LUIZ H. C.; ANDRADE-SILVA, DEBORA; SHIBURAH, MARK E.; DE OLIVEIRA, BEATRIZ C. D.; PAIVA, STEPHANY C.; ABUCHERY, BRYAN E.; FERRI, YETE G.; FONTES, VERONICA S.; DE OLIVEIRA, LEILANE S.; DA SILVA, MARCELO S.; et al. Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?. CELLS, v. 10, n. 11, . (19/10753-2, 20/08162-3, 21/04253-7, 20/00316-1, 20/16465-6, 18/04375-2, 21/05523-8, 20/10277-3, 19/25985-6, 20/16480-5)

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