Optimization of the CRISPR-Cas9 gene deletion system in macrophages and evaluation of genes potentially involved in NLRC4 inflammasome activation

Author(s)

Letícia de Sousa Lopes

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-08-16

Examining board members

Dario Simões Zamboni; Elaine Zayas Marcelino; Tiago Wilson Patriarca Mineo

• Advisor: Dario Simões Zamboni; Warrison Athanásio Coelho de Andrade

Abstract

The inflammasome is a multiprotein complex involved in the activation of inflammatory caspases in response to environmental signals or pathogens. After ligand recognition, the inflammasome complex is formed and mediates the cleavage and activation of pro-IL-1β and pro-IL-18, as well as gasdermin-D, leading to the secretion of cytokines in their active forms and inflammatory cell death, known as pyroptosis. Among the different inflammasomes, NLRC4 and NLRP3 are the most studied. The NLRC4 inflammasome plays a crucial role in host defense against intracellular pathogens, including Salmonella, Shigella, Legionella and Pseudomonas. Furthermore, mutations in the NLRC4 gene have been described to be linked to the onset and progression of inflammatory diseases such as MAS (Macrophage Activation Syndrome), and neonatal onset enterocolitis. Despite being one of the most studied inflammasomes, mechanisms involved in the activation and regulation of the NLRC4 pathway have yet to be identified. In this work, we optimized the CRISPR-Cas9 technique in bone marrow-derived macrophages for the deletion of genes previously identified in our laboratory, in order to evaluate a possible role in NLRC4 inflammasome activation in response to Legionella pneumophila infection. Throughout this project we were able to standardize and optimize the gene deletion protocol by CRISPR Cas9, generating primary bone marrow-derived macrophages nocautes for ASC and NLRC4 genes. Furthermore, we observed that the possible deletion of our genes of interest, did not lead to increased bacterial replication, suggesting that the deleted proteins do not seem to play a relevant role in the functions of the NLRC4 inflammasome. (AU)

• FAPESP's process: 20/06177-3 - Optimization of a CRISPR/Cas9 genetic deletion system in macrophages to evaluate proteins important for the activation of NLRC4 inflammasome in macrophages
• Grantee: Letícia de Sousa Lopes
• Support Opportunities: Scholarships in Brazil - Master