Identification of critical cellular factors during neurotropic arbovirus infection through global screening with CRISPR-Cas9 libraries

• Grant number: 22/11885-2
• Support Opportunities: Research Grants - Visiting Researcher Grant - Brazil
• Start date: March 01, 2023
• End date: February 29, 2024
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Thiago Mattar Cunha
• Grantee: Thiago Mattar Cunha
• Visiting researcher: Rafael Freitas de Oliveira Franca
• Visiting researcher institution: Ministério da Saúde (Brasil). Fundação Oswaldo Cruz (Fiocruz), Brazil
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 13/08216-2 - CRID - Center for Research in Inflammatory Diseases, AP.CEPID

Abstract

Arboviruses represent a serious public health problem which hasWorsened in recent years due to the insertion of new viruses into highlysusceptible. The concomitant circulation of different viruses results in a lower load thanhealth systems, which is unprepared to deal with the different clinical formsthese infections. It is known that arbovirus infection usually results in a February frameacute, however a significant portion of infections for the developmentframes communicated graves. In this context, molecules that do not participatecontrol of evolution versus evolution of unknown forms.The present proposal aims to evaluate the applicability of a CRISPR-Cas9 library as a toolfor the identification of critical factors for infection and cellular damage caused byarbovirus of interest. This library consists of grouping 77,441 targeted tabsfor the deletion of approximately 19,000 human genes. In this way, mainlya global scan of the human genome for factors related to the response of theagainst experimental arbovirus infection, through gene deletion mediated byCRISPR/Cas9 in permissive cells. After the construction of cell clones expressing theconstitutively Cas9 enzyme and library transduction by lentiviruses, sub-meters asknockout cell populations to lethal infection with Zika, Chikungunya andMayaro. Finally, to determine genes and pathways involved in cell replication and damage throughof the sequencing of the surviving cells and identification of the guides present at that timepopulation. The identified genes were ranked and grouped according to the pathway orcellular process which participates through genetic annotation tools (Gene Ontology).Functional assays for the identified genes will be carried out later, which may include,but not limited to: i) viral load assays for receptor-coding genesand/or surface proteins ii) cell death assays for genes involved in the evaluationsignaling for apoptosis or other forms of cell death iii) cytokine and chemokine profileinflame to genes involved in inflammation. In this way, a giftproposal may contribute to the identification of molecular pathways in the infection byarbovirus, which can add new knowledge about the interaction processeshost virus, which in turn can lead to the discovery of new targetstherapeutic. (AU)