目的 利用规律间隔成簇短回文重复序列关联蛋白9（CRISPR/Cas9）技术构建刚地弓形虫（Toxoplasma gondii）类枯草杆菌蛋白酶基因sub3敲除株并进行体外表型分析，探究TgSUB3对弓形虫黏附、入侵和生长繁殖的作用。 方法 利用定点突变方法将pSAG1::CAS9-U6::sgUPRT中的sgUPRT突变为Tgsub3的单向导RNA（sgRNA），构建sub3基因敲除质粒pSAG1::CAS9-U6::sgSUB3。PCR扩增带有sub3上下游40 bp同源臂的二氢叶酸还原酶抗性供体片段，将质粒和供体片段通过电转化的方式导入弓形虫RH∆ku80速殖子，经过乙胺嘧啶抗性选择和单克隆筛选，PCR鉴定敲除株RH∆ku80∆sub3（∆sub3）。鉴定正确的敲除株∆sub3进行噬斑试验、入侵试验和增殖试验，分析其体外表型，以RH∆ku80为对照，使用软件 GraphPad Prism 9进行统计学分析。 结果 经PCR鉴定，在∆sub3中扩增到的条带大小符合预期，∆sub3敲除株构建成功。噬斑试验结果显示，RH∆ku80和∆sub3形成的噬斑面积分别为（60.42 ± 23.20）任意单位和（2.21 ± 1.89）任意单位，二者差异有统计学意义（t = 17.79，P < 0.01）；入侵实验结果显示，RH∆ku80和∆sub3的入侵效率分别为（37.94 ± 18.18）%和（22.97 ± 15.36）%，差异无统计学意义（t = 0.89，P > 0.05）。增殖试验结果显示，RH∆ku80和∆sub3纳虫泡中含有8个及以上速殖子的纳虫泡数量分别为 (56.33 ± 8.58）% 和 (39.67 ± 11.84）%；含有4个及以下速殖子的纳虫泡数量分别为（43.67 ± 8.58）%和（60.33 ± 11.84）%，与 RH∆ku80 虫株相比，纳虫泡内速殖子个数显著减少（F = 17.93，P < 0.01）。 结论 成功构建弓形虫∆sub3敲除株，TgSUB3的缺失影响弓形虫速殖子的生长繁殖。

Objective To generate the Toxoplasma gondii sub3 (Tgsub3) gene knockout strain using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, investigate the in-vitro phenotypes of the Tgsub3 gene knockout strain, and examine the effect of Tgsub3 gene on adhesion, invasion and proliferation of T. gondii. Methods SgUPRT on the pSAG1::CAS9U6::SgUPRT plasmid was mutated to single guide RNA (sgRNA) using site mutation, and the pSAG1::CAS9-U6::sgSUB3 plasmid with the sub3 gene knockout was generated. The DHFR resistant donor fragments containing 40 bp upstream and downstream homology arms of the sub3 gene were amplified, and the sub3 gene knockout plasmid and donor fragments were co-transfected into T. gondii by electroporation. Following resistance selection by pyrimethamine and monoclonal screening, the sub3 gene knockout strain RH∆ku80∆sub3 (∆sub3) was identified using PCR assay. The in-vitro phenotypes of the ∆sub3 strain were analyzed with plaque, invasion, and proliferation assays. Using RH∆ku80 strain as a control, all statistical analyses were conducted using the software GraphPad Prism 9. Results PCR assay identified bands with expected sizes in the ∆sub3 strain, indicating successful generation of the ∆sub3 strain. Plaque assay showed that the sizes of plaques formed by RH∆ku80 and ∆sub3 strains were (60.42 ± 23.20) au and (2.21 ± 1.89) arbitrary unit, respectively (t = 17.79, P < 0.01), and invasion assay showed that the invasion efficiencies of RH∆ku80 and ∆sub3 strains were (37.94 ± 18.18) % and (22.97 ± 15.36) %, respectively (t = 0.89, P > 0.05). Proliferation assay showed that the proportions of parasitophorous vacuoles containing 8 and more tachyzoites of RH∆ku80 and ∆sub3 strainswere (56.33 ± 8.58) % and (39.67 ± 11.84) %, respectively, and the proportions of parasitophorous vacuoles containing 4 and fewer tachyzoites of RH∆ku80 and ∆sub3 strains were (43.67 ± 8.58) % and (60.33 ± 11.84) %, respectively. Compared with control strain, the number of tachyzoites within the parasitophorous vacuole was significantly decreased (F = 17.93, P < 0.01). Conclusion The ∆sub3 gene knockout strain is successfully generatedand absence of the Tgsub3 gene affects the growth and reproduction of T. gondii tachyzoites.