The Application Of TALEN And CRISPR Systems In The Study Of Pathogenic Mechanisms Of Toxoplasma Gondii

With the development of molecular biology techniques, scientists have gained more and more insights into the detailed functions of genetic materials, benefiting from the improvements of genome engineering tools. DNA targeting technology experienced several major changes from ZFN to TALEN then to CRISPR in the last eight years, which also resulted in breakthroughs to the research on functional genomics of Apicomplexan parasites, especially Toxoplasma gondii. T. gondii is an obligate intracellular parasite, which causes serious zoonosis to the reproductive health of human and the development of livestock. Although significant achievements have been made towards the understanding of T. gondii biology, the mechanisms about its pathogenesis are still poorly understood. The emerging genetic technologies provide good opportunities to address some of these questions.In this study, we first used TALEN to inactivate RON2, which plays an important role during invasion. Then individually tested the efficacy of three functional domains of RON2 as DNA vaccines. Subsequently used the CRISPR-Cas9 system to knock out GRA7, ROP17, ROP18 in a local strain T.g HB1 to test their contribution to the virulence in mice. In addition, we expanded the application of the CRISPR-Cas9 system and designed an approach to use it for regulation of gene expression in T. gondii.1. TALEN mediated inactivation of RON2 and immunoprotection of RON2 based DNA vaccines.According to the genomic sequence of RON2, TALEN plasmids were designed and constructed to target RON2. After transfection of TALEN plasmids, the positive clones were selected based on their GFP and RFP expression. RON2 disruptions were detectable in the pool by PCR but we failed to get viable clones, suggesting the essentiality of this gene. Based on this, we were interested in RON2 as a vaccine candidate. RON2 were truncated into three fragments and individually cloned into the vector pc DNA3.1 to construct the plasmids pc DNA3.1-RON2-1/2/3. To test their protection as DNA vaccines, 100 μg recombinant plasmid was given to each mouse by intramuscular injection at 0 d, 14 d, 28 d and 42 d respectively, the vector and PBS were used as controls. After vaccination, ELISA was used to check the cytokine production from mice and showed that all RON2 fragments induced the high levels of IFN-γ. Subsequent parasite challenging experiments indicated that only RON2-3 offered a 16.7% protection rate, which suggest that the D3 and D4 domains of RON2 outside of host cell membrane might be a good candidate for vaccine.2. Deletion of virulence factors in T.g HB1 by the CRISPR-Cas9 system.At first, we constructed CRISPR-Cas9 plasmids to target GRA7, ROP17, ROP18 respectively and homology templates that were used to replace the coding sequences of corresponding genes with the selectable markers(GFP, DHFR* or CAT). Then co-transfected the CRISPR plasmid and homology template into parasites. After selection, diagnostic PCR and Western-blotting were used to screen and check the knockout clones. Through approach, we deleted GRA7, ROP17, ROP18 individually and in different combinations. Subsequent plaque assay suggested that all the mutants had no obviously growth defects in vitro. Animal challenge experiment showed that the single deletion mutants △GRA7, △ROP17, △ROP18 in T.g HB1 strain had different attenuation in virulence, while T.g HB1△GRA7△ROP18 displayed a partial attenuation like the T.g HB1△ROP18 mutant, same with T.g HB1△GRA7△ROP17 and T.g HB1△ROP17. These results indicated that GRA7, ROP17, ROP18 played important roles in virulence determination in genetical diverse strains.3. CRISPR-d Cas9 mediated gene expression regulation in Toxoplasma gondii.Replaced UPRT with SAG1P::RFP-SAG13’ by CRISPR-Cas9 to construct a parasite line expressing RFP driven by the SAG1 promoter at the UPRT locus; then, modified the CRISPR-Cas9 system by mutating the Cas9 to block its nuclease activity(d CAS9) and fused it with transcription regulators KRAB. Then directed the d CAS9-KRAB to different parts of SAG1 promoter by introducing different sg RNAs. Transfected then into UPRT::SAG1P::RFP-SAG13’ parasites to examine the control of RFP expression. The results indicated that sg RNA targeting-316 to-297 bp of the SAG1 promoter led to a maximum expression of endogenous SAG1 and RFP using d CAS9-KRAB fusion.In this study, we mutated RON2 by TALEN and confirmed that RON2 was essential for parasite survival; also verified that RON2 might be a good vaccine candidate. Using the CRISPR-Cas9 system knocking out corresponding genes, we checked the contribution of GRA7, ROP17, ROP18 to the virulence of T.g HB1 in mice, the results of which suggested the genetic diversity between T.g HB1 and RH. Finally, we tested the potential application of CRISPR-d Cas9 to regulate gene regulation in Toxoplasma gondii.