Laboratory of Medical Zoology, Department of Medicine, Mie University, Mie, Japan.
Department of Environmental Parasitology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Sci Rep. 2021 Sep 16;11(1):18501. doi: 10.1038/s41598-021-97984-z.
The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.
CRISPR/Cas9 系统是一种强大的疟原虫基因工程技术。我们在此报告通过将表达 Cas9 的寄生虫与线性供体模板 DNA 相结合进一步改进 CRISPR/Cas9 系统。通过双交叉重组将 cas9 基因插入基因组中产生表达 Cas9 的寄生虫。通过将线性供体模板和携带 sgRNA 的质粒转染表达 Cas9 的寄生虫裂殖子，在两周内高效（>85%）实现定点突变和荧光蛋白融合。值得注意的是，在产生的转基因寄生虫中没有脱靶突变，也没有意外重组，这是当前 CRISPR/Cas9 系统的技术问题。此外，使用我们的系统，在单次转染中成功修饰了两条位于不同染色体上的基因。由于其高效性和稳健性，我们改进的 CRISPR/Cas9 系统将成为疟原虫基因工程的标准技术，这将极大地推进对该寄生虫的未来研究。
