Genome engineering of Toxoplasma gondii using CRISPR/Cas system

• Main Author: Muhammad Hanif Zaini
• Other Authors: Ajai Vyas
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/63633
• Institution: Nanyang Technological University

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Behavioural change from aversion to attraction towards cat odour have been observed in Toxoplasma gondii infected rodents. Anti-psychotic drugs haloperidol and valproic acid as well as dopamine intake inhibitor GBR12909 were observed to rescue this behavioural change in Toxoplasma gondii infected rodents. Thus alterations in dopamine biosynthesis pathway have been proposed as the underlying mechanism behind these behavioural modifications due to the parasite-encoded tyrosine hydroxylase genes, TgAAH1 and TgAAH2. Tyrosine hydroxylase is the rate limiting enzyme in dopamine biosynthesis and responsible for the production of dopamine’s precursor, L-DOPA. Therefore we intend to generate mutant Toxoplasma gondii by knocking out TgAAH1 and TgAAH2 genes using CRISPR/Cas system. CRISPR/Cas plasmid, pU6-Protospacer-1, targeting the N-termini sequence of TgAAH1 and TgAAH2 genes was successfully constructed. In addition, the plasmid was also successfully transfected into Toxoplasma gondii using PEI transfection. Efficacy of pU6-Protospacer-1 in introducing insertions or deletions of nucleotides (indels) into the target site by NHEJ have yet to be determined as experiments are still underway in isolating and subsequent sequencing of single clones of transfected Toxoplasma parasites.