Evaluation of CRISPR-Cas9 delivery methods in THP-1 cell lines for genomic editing targeting PPAR± gene

• Grant number: 18/23089-0
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Effective date (Start): March 01, 2019
• Effective date (End): August 31, 2019
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Paulo Eduardo Martins Ribolla
• Grantee: Isabela Gaseta Ferraz Paiva
• Supervisor: Aebischer Anton
• Host Institution: Instituto de Biotecnologia (IBTEC). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Research place: Robert Koch Institute (RKI), Germany
• Associated to the scholarship: 17/25854-3 - Evaluation of the allelic variant (L162V) of the human PPARalfa gene in the establishment of Leishmania infantum infection: use of CRISPR-Cas9 technology for knock-in in THP-1 cell line, BP.MS

Abstract

Peroxisome Proliferator Activated Receptor alpha (PPAR±) was shown as a possible candidate gene to contribute to the genetically determined risk of Visceral Leishmaniasis (VL). In a case-control study conducted by our group in the VL endemic area of Teresina-PI-Brazil, genotypes containing the mutated 162V allele were significantly more frequent among individuals with VL than among all uninfected individuals. The relationship of Leishmania infantum infection with serum lipid levels and lipid modulation by PPAR± suggests that biological assays with allelic variants of these receptors on L. infantum-infected macrophages may represent a good way forward for understanding parasite-host interaction in the VL. In addition, novel genetic manipulation techniques, such as CRISPR-Cas9, associated with stable cell lines with high proliferative capacity allow the introduction of alleles of interest (in the case of the PPAR± gene, the 162V allele) and subsequent screening with the use of selectable markers in the application in functional studies for the evaluation of host cell target genes (macrophages) in L. infantum infection.Despite the great promise of the CRISPR-Cas9 genome-editing system, several challenges remain to be tackled before its successful applications for human patients. The biggest challenge is the safe and efficient delivery of the system to target cells. In this project we propose to test different types of delivery, focusing in Electroporation versus Lipofection. (AU)