目的 基于成簇规律间隔短回文重复序列核酸酶技术（CRISPR/Cas9）构建刚地弓形虫ME49株缓殖子形成缺陷基因2（bfd2）缺陷虫株并进行表型分析，探讨弓形虫BFD2对虫体分化和增殖作用。方法 运用E-CRISPR数据库设计bfd2向导RNA（SgRNA），并使用Q5定点基因敲除试剂盒突变pSAG1::CAS9-U6::SgUPRT质粒上的SgRNA，构建pSAG1::CAS9-U6::SgBFD2质粒。将乙胺嘧啶抗性基因、bfd2基因上下游序列连接成供体DNA（donorDNA）并克隆于pUC19质粒上，PCR扩增donorDNA。以ME49虫株为背景，pSAG1::CAS9-U6::SgBFD2质粒和donorDNA片段电穿孔共转染ME49弓形虫速殖子，电转后悬液接种至人包皮成纤维细胞（HFF），3 μmol/L乙胺嘧啶单克隆筛选电转虫株。将筛选出的单克隆虫株接种HFF细胞，培养传代后，PCR鉴定虫株∆bfd2敲除情况。通过体外增殖试验观察虫株在HFF内的增殖情况，噬斑试验观察虫株在HFF细胞上形成噬斑的能力，苏木精-伊红（HE）染色观察虫株感染小鼠的肝脏病理情况。采用GraphPad Prism 9进行统计学分析，实验数据使用双尾非成对Student t检验和ANOVA检验。结果 成功构建pSAG1::CAS9-U6::SgBFD2质粒和donorDNA质粒，编码乙胺嘧啶抗性基因（DHFR）序列成功插入靶位点，经PCR鉴定、筛选出∆bfd2基因敲除单克隆虫株（ME49 ∆bfd2株）。增殖试验结果显示，ME49株感染的HFF内，每个纳虫泡中 > 16、= 16和 ≤ 8个速殖子的纳虫泡数量占比分别为（55.33 ± 5.03）%、（27.00 ± 3.00）%和（17.67 ± 4.04）%，ME49 ∆bfd2株感染的分别为（25.33 ± 4.16）%、（42.67 ± 3.06）%和（32.00 ± 6.93）%，ME49株感染的HFF中 > 16个速殖子的纳虫泡数量高于ME49 ∆bfd2虫株（t = 6.337，P < 0.01）。噬斑试验结果显示，接种100、1 000、10 000个ME49株速殖子感染的HFF分别形成（13.50 ± 3.11）、（119.75 ± 4.86）和（264.25 ± 28.61）个噬斑，均高于ME49 ∆bfd2株感染的（1.25 ± 0.96）、（6.75 ± 0.96）和（22.00 ± 5.72）个（t = 7.415、57.72、18.04，均P < 0.01）。HE结果显示，ME49 ∆bfd2株感染小鼠肝脏炎症较ME49株感染小鼠减轻。结论 基于CRISPR/Cas9技术成功构建BFD2缺陷虫株，且BFD2的缺失可抑制弓形虫的分化、增殖，并减轻小鼠肝脏炎症。

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.