Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Praha, Czech Republic.
Department of Microbiology and Molecular Genetics, College of Biological Sciences, UC Davis, Davis, CA, USA.
Open Biol. 2022 Apr;12(4):210361. doi: 10.1098/rsob.210361. Epub 2022 Apr 27.
CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. , a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the assembled CRISPR/Cas9 components to successfully edit the genome of . The cell line that stably expresses Cas9 in both nuclei of showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, , and The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in .
CRISPR/Cas9 介导的基因组编辑已成为一种非常强大的技术，可用于修饰许多生物体中的基因表达，包括寄生原生动物。这种原生动物寄生虫每年在全球感染约 2.8 亿人，由于其四倍体基因组，一直无法使用 CRISPR/Cas9 来生成敲除细胞系。在这项工作中，我们展示了 组装的 CRISPR/Cas9 组件成功编辑 基因组的能力。在 的两个核中稳定表达 Cas9 的细胞系显示出对包含 gRNA 和抗性标记转录单元的盒的有效重组。这个高效的过程导致三个独立实验基因 、 和 的所有基因拷贝一次性被删除。该方法也适用于必需基因的不完全破坏，如 的表达显著降低所证明的那样。最后，测试 Cas9 诱导的重组的效率表明，同源臂短至 150bp 就足以在 中建立一个完整的敲除细胞系。
