Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9.
Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9.
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使用 CRISPR/Cas9 标记恶性疟原虫的内源基因
DOI：
10.1186/s13071-017-2539-0
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发表时间：
2017-12-02
影响因子：
3.2
通讯作者：
Dai X
中科院分区：
文献类型：
--
作者：
Kuang D;Qiao J;Li Z;Wang W;Xia H;Jiang L;Dai J;Fang Q;Dai X
背景：恶性疟原虫是最致命的疟疾寄生虫。目前，针对恶性疟原虫蛋白的商品化抗体很少，这极大地限制了对恶性疟原虫的研究。CRISPR/Cas9是一种高效的基因组编辑方法，已被广泛应用于各种生物体中。然而，这项技术在恶性疟原虫中的应用仍然局限于基因敲除、定点突变和绿色荧光蛋白(GFP)报告系的产生以及插入位点的中断。为了在CK2β1、CK2α和StK上添加HA或HA-Ty1标签，构建了含有标签序列、特定同源臂和sgRNA的pL6cs-hDHFR-CK2β1/CK2α/StK。用电穿孔法将pUF1-BSD-Cas9和pL6cs-hDHFR-CK2β1/CK2α/StK质粒导入恶性疟原虫3D7株。之后，将BSD和WR99210药物加入培养物中，以筛选含有这两种质粒的寄生虫。电穿孔后20天，出现活体寄生虫，收集标本进行聚合酶链式反应、DNA测序、Western blotting和免疫荧光检测。结论：改进了CRISPR/Cas9方法整合恶性疟原虫基因标签的方法，为在分子水平上进一步研究恶性疟原虫奠定了基础。
Background:Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. However, the use of this technique in P. falciparum is still limited to gene knockout, site-specific mutation and generation of green fluorescent protein (GFP) reporter line with disruption of inserted sites.Results:We have adapted the CRISPR/Cas9 system to add commercial tag sequences to endogenous genes of P. falciparum. To add HA or HA-TY1 tags to ck2β1, ck2α and stk, pL6cs-hDHFR-ck2β1/ck2α/stk was constructed, which contained sequences of tags, specific homologous arms, and sgRNA. The P. falciparum 3D7 strain was subsequently transfected with pUF1-BSD-Cas9 and pL6cs-hDHFR-ck2β1/ck2α/stk plasmids via electroporation. After that, BSD and WR99210 drugs were added to the culture to screen parasites containing both plasmids. Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins.Conclusions:We have improved the method to integrate tags to Plasmodium falciparum genes using the CRISPR/Cas9 method, which lays the foundation for further study of Plasmodium falciparum at the molecular level.
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影响因子：
48
作者：
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通讯作者：
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影响因子：
3.1
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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