Division of Infectious Diseases, Department of Internal Medicine, Stanford University, Grant Building, S-143, 300 Pasteur Drive, Stanford, CA 94305, United States.
Division of Infectious Diseases, Department of Internal Medicine, Stanford University, Grant Building, S-143, 300 Pasteur Drive, Stanford, CA 94305, United States; Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, United States.
Int J Parasitol. 2021 Feb;51(2-3):193-200. doi: 10.1016/j.ijpara.2020.09.005. Epub 2020 Nov 29.
The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimised CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilising domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.
原虫寄生虫溶组织内阿米巴是一种重要的人类病原体，也是全球范围内寄生虫致死的主要原因。缺乏用于基因组编辑的分子工具，阻碍了对这种寄生虫重要生物学功能的研究。由于其多功能性，CRISPR（成簇的规律间隔的短回文重复）-Cas9 系统已成功用于诱导特定基因组改变，包括原虫寄生虫。在这项研究中，我们优化了 CRISPR-Cas9，将其用作溶组织内阿米巴的遗传工具。我们选择了一种包含指导 RNA（gRNA）和 Cas9 核酸酶表达盒的单质粒方法。使用阿米巴 U6 启动子驱动 gRNA 的表达，并通过 Northern blot 分析证实其表达。通过与 myc 标记的 Cas9（ddCas9）融合的二氢叶酸还原酶的不稳定结构域，获得稳定转染的细胞系。使用小分子配体三甲氧苄氨嘧啶（TMP）处理 16 小时后，我们能够诱导 ddCas9 的表达。表达 ddCas9 和 Luc-gRNA 或非特异性（NS）-gRNA 的稳定细胞系用含有突变荧光素酶基因（pDeadLuc）的质粒瞬时转染，该基因由 Luc-gRNA 靶向，并用含有截断荧光素酶基因（pDonorLuc）的另一个质粒恢复荧光素酶表达和随后的活性。我们观察到表达 Luc-gRNA 的细胞系的发光信号增加，这表明 Cas9 活性促进了同源重组。PCR 检测到嵌合 DNA 的存在以及通过同源重组获得的修复 DNA 的测序结果证实了这一点。我们相信这是首次报道溶组织内阿米巴中使用 CRISPR/Cas9 系统，并提供了证据表明，这种基因组编辑方法可用于这种早期分支真核生物的遗传研究。
