利用CRISPR/Cas9快速制备疟原虫基因修饰模型的研究

Malaria killed about one million people annually. The ability to precisely and efficiently modify genome is critical to fully elucidate the gene function in Plasmodium yoelli and Plasmodium falciparum, which are the common used parasite models in malaria study. However, we possessed poor choices of effective method performing genome editing, which limits the gene functional elucidation. As an emerging efficient tool, clustered, regularly interspersed short palindromic repeats (CRISPR) /Cas9 has been successfully applied in genome editing at a wide range of model organisms. Two key effector in this system, nuclease Cas9 and guide RNA sgRNA, form Cas9/sgRNA complex to perform the targeted DNA recognition and double strand break. Here we report a method to engineer Plasmodium genome using the CRISPR RNA-guided Cas9 nuclease and homologous recombination. In recent work, we have established the expression and nuclear localization of Cas9 driven by 2A peptide with hDHFR selector marker in P.yoelli. U6 snRNA promoter was cloned and used for driven transcript expression of sgRNA in P.yoelli. 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, reporter or tag addition, and nucleotide replacement. Furthermore, genomic modifications in multiple loci were generated by sequential targeting strategy without drug-selector marker gene integration. Our results establish a methodology to produce any desired modification in the genome of P.yoelli and P.falciparum at low cost. This technology is an important improvement to the limited genetic techniques available so far in this model organism.

疟疾每年导致全球近百万人口的死亡，其致病寄生虫疟原虫的基因功能研究至今缺乏高效工具。CRISPR/Cas9作为新型高效基因组修饰方法，成功应用于多种模式物种。我们前期工作建立了CRISPR系统的效应分子核酸酶Cas9在约氏疟原虫细胞的异源表达和细胞核定位；克隆了约氏疟原虫U6 snRNA启动子，并且能够驱动另一个效应分子sgRNA转录表达；构建了基于同源重组修复的Cas9/sgRNA/Donor载体系统，成功实现约氏疟原虫基因不同类型修饰，制备出内源基因Sera1和Sera2的基因敲除模型；内源基因Sep3-GFP和Sep3-Myc标签插入模型和内源基因Hsp70核苷酸替换模型。本研究将在疟疾生物学最常用的两种模型（鼠约氏疟原虫和人恶性疟原虫）建立CRISPR/Cas9基因组修饰方法，快速精确制备基因敲除、标签插入和基因替换修饰模型，为疟原虫基因功能揭示提供高效工具。

该项目建立了鼠约氏疟原虫的CRISPR/Cas9基因组修饰方法，能够高效进行疟原虫的基因敲除、基因加标签和核苷酸替换修饰；在此工作的基础上，针对疟原 虫基因功能研究中多基因修饰的技术需求，进一步开发了多基因修饰的方法。结合耐药标记基因的负筛选和顺序修饰，能够在疟原虫模型中实现多重修饰；利用Cas9方法，该项目对鼠约氏疟原虫的AP2家族的26个成员基因开展了基因敲除和基因标签插入，系统分析了疟原虫AP2转录因子的功能，为理解疟原虫生活周期中的基因表达调控提供了一幅“全景图”。