伯氏疟原虫Cas9基因组编辑工具的研究

项目名称： 伯氏疟原虫Cas9基因组编辑工具的研究

项目编号： No.31501912

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 动物学

项目作者： 崔慧婷

作者单位： 厦门大学

项目金额： 20万元

中文摘要： 伯氏疟原虫是疟疾生物学研究的重要模型，然而其基因功能研究缺乏高效工具。我们之前的工作已经建立约氏疟原虫CRISPR/Cas9基因组修饰方法，然而在伯氏疟原虫是否有效仍然未知。本课题的前期工作建立了外源核酸酶Cas9在伯氏疟原虫细胞的细胞核表达和定位；验证了约氏疟原虫U6 snRNA启动子能够驱动sgRNA在伯氏疟原虫细胞中转录表达；构建了基于同源重组修复的Cas9/sgRNA/Donor质粒载体系统。进一步，我们在伯氏疟原虫ANKA虫株中完成了不同类型基因组修饰测试，制备出内源基因Pbmif和Pbap2-g的基因敲除模型；内源基因Pbmif-Myc标签插入模型和内源基因Pbmif核苷酸替换模型。本研究将在伯氏疟原虫中建立CRISPR/Cas9基因组修饰工具，为疟疾生物学研究提供高效研究工具。

中文关键词： 疟疾；伯氏疟原虫；基因编辑；同源重组；CRISPR/Cas9

英文摘要： Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. As an emerging method, clustered regularly interspersed short palindromic repeats (CRISPR) /Cas9 has been applied in genome editing at may model organisms. Two key components in this system, nuclease Cas9 and guide RNA sgRNA, form Cas9/sgRNA complex to perform the targeted DNA recognition and double strand break. Our previously work has established the CRISPR/Cas9-meditaed genome editing method in a rodent malaria parasite Plasmodium yoelii. However, does this method perform well in another rodent malaria parasite model Plasmodium berghei is unknown? In recent work, we have established the expression and nuclear localization of Cas9 driven by 2A peptide with hDHFR selector marker in P. berghei. U6 snRNA promoter derived from P.yoelii is capable of driving transcription of sgRNA in P. berghei. We further demonstrate that Cas9/sgRNA is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, tag addition, and nucleotide replacement targeting to endogenous gene in P. berghei. This study is to establish and optimize the genome editing method based on Cas9/sgRNA system in P. berghei. This technology is an important improvement to the limited genetic techniques available so far in this model organism.

英文关键词： Malaria;Plasmodium berghei;Genome editing;Homologous recombination;CRISPR/Cas9