关键词: 刚地弓形虫, 缓殖子形成缺陷基因2, CRISPR/Cas9, 表型分析, 质粒

Abstract:

Objective To generate the bradyzoite formation deficiency 2 (bfd2) gene-deficient Toxoplasma gondii ME49 strain based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system, analyze the phenotype of the strain, and investigate the effect of bfd2 on T. gondii differentiation and proliferation. Methods Small guide RNA (sgRNA) for bfd2 was designed using the E-CRISPR tool, and the sgRNA in the pSAG1::CAS9-U6::SgUPRT plasmid was mutated using the Q5 Site-Directed Mutagenesis Kit to generate the pSAG1::CAS9-U6::SgBFD2 plasmid. The pyrimethamine resistance gene and the upstream and downstream sequences of the bfd2 gene were ligated to form donor DNA and cloned into the pUC19 plasmid, and donor DNA was amplified using PCR assay. The pSAG1::CAS9-U6::SgBFD2 plasmid and donor DNA fragments were electroporation co-transfected into tachyzoites of the T. gondii ME49 strain. Following electroporation, the suspension was inoculated into human foreskin fibroblasts (HFFs), and the electroporated parasite strains were subjected to monoclonal selection with 3 μmol/L pyrimethamine. The screened monoclonal parasite strains were inoculated into HFF cells and passaged, and the Δbfd2 knockout efficiency in the strain was checked using PCR assay. The proliferation of the strains was measured in HFF cells using in vitro proliferation assays, and the plaque-forming ability of the strain was tested in HFFs using plaque assays. In addition, the pathological changes in livers of mice infected with the strains were observed using hematoxylin and eosin (HE) staining. All statistical analyses were performed using the software GraphPad Prism 9, and the experimental data was tested for statistical significance using two-tailed non-paired Student t-test and analysis of variance (ANOVA). Results The pSAG1::CAS9-U6::SgBFD2 and donor DNA plasmid were successfully generated. The dihydrofolate reductase (DHFR)-coding sequence was successfully inserted to the target site, and the monoclonal ME49 ∆bfd2 gene knockout strain (ME49 ∆bfd2 strain) was successfully screened as verified by PCR assay. In vitro proliferation assays revealed that the proportions of parasitophorous vacuoles containing more than 16, 16 and 8 and less tachyzoites were (55.33 ± 5.03)%, (27.00 ± 3.00)% and (17.67 ± 4.04)% in HFF cells inoculated with the ME49 strain, and (25.33 ± 4.16)%, (42.67 ± 3.06)% and (32.00 ± 6.93)% in HFF cells inoculated with the ME49 ∆bfd2 strain, and the number of parasitophorous vacuoles containing more than 16 tachyzoites was higher in HFF cells infected with the ME49 strain than in cells infected with the ME49 ∆bfd2 strain (t = 6.337, P < 0.01). Plaque assays showed (13.50 ± 3.11), (119.75 ± 4.86) and (264.25 ± 28.61) plaques in HFFs inoculated with 100, 1 000 and 10 000 tachyzoites of the ME49 strain, and (1.25 ± 0.96), (6.75 ± 0.96) and (22.00 ± 5.72) plaques in HFFs inoculated with 100, 1 000 and 10 000 tachyzoites of the ME49 ∆bfd2 strain (t = 7.415, 57.72, 18.04, all P < 0.01). HE staining showed alleviation of liver inflammation in mice infected with the ME49 ∆bfd2 strain relative to in mice infected with the ME49 strain. Conclusion The BFD2 deficient strain of T. gondii has been successfully generated using the CRISPR/Cas9 system, and BFD2 deficiency inhibits T. gondii differentiation and proliferation and alleviates liver inflammation in mice.

Key words: Toxoplasma gondii, Bradyzoite formation deficient 2, CRISPR/Cas9, Phenotype analysis, Plasmid