CRISPR/Cas9 technology application in L. amazonensis gene editing: production of knockouts and tagging of enzymes involved in L-arginine metabolism.

• Grant number: 23/07024-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2023
• End date: November 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Lucile Maria Floeter-Winter
• Grantee: Vinícius Yamazaki Gomes
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 18/23512-0 - The Leishmania-host relationship from the ‘omics’ perspective, AP.TEM

Abstract

Organisms of the genus Leishmania are obligate intracellular parasites of cells of the mononuclear phagocytic system of many vertebrate hosts. Their biology offers unique characteristics as a study model in the pathogen-host interaction.Specifically, the proposal submitted here focuses on the AAP3 transporter. The application of CRISPR/Cas9 technology has been used in the Laboratory's projects and has generated interesting results in the characterization of L-arginine metabolism targets in L. amazonensis. The strain expressing the Cas9 endonuclease (La-Cas9) is well established and has been used to generate knockout mutants of the L-arginine amino acid permease transporter 3 (aap3). However, as already presented in previous reports of the thematic project in question, the coding sequence of this transporter is present in two tandem copies, in a tetraploid chromosome. This characteristic makes it technically difficult to obtain knockout strains and some strategies are being considered to overcome the imposed difficulties.In this project it is proposed to obtain L. amazonensis null knockout mutants of AAP3 by applying the CRISPR/Cas9 protocol, with changes in the initially proposed protocol, in order to use guide RNAs that direct the endonuclease to sequences adjacent to the two copies of the gene. Among the strategic changes are the use of alternative regions as cleavage targets, in addition to obtaining a background lineage that expresses the protein to be deleted through an episome. More reliable sequences than those obtained from public databases were obtained in the meantime, with the sequencing of cosmids from the genomic library of the L. amazonensis strain used in our studies. This allows us to design guide RNA templates more safely and invest in new attempts to fully delete our target.