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Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept
International Journal for Parasitology ( IF 4 ) Pub Date : 2020-11-29 , DOI: 10.1016/j.ijpara.2020.09.005
Monica Mendes Kangussu-Marcolino 1 , Pedro Morgado 1 , Dipak Manna 1 , Heather Yee 1 , Upinder Singh 2
Affiliation  

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.



中文翻译:

在溶组织内阿米巴中开发 CRISPR/Cas9 系统:概念验证

原生动物寄生虫溶组织内阿米巴是一种重要的人类病原体,也是全球范围内主要的寄生虫死亡原因。缺乏用于基因组编辑的分子工具阻碍了对该寄生虫重要生物学功能的研究。由于其多功能性,CRISPR(成簇的定期间隔短回文重复序列)-Cas9 系统已成功用于诱导位点特异性基因组改变,包括原生动物寄生虫。在这项研究中,我们优化了 CRISPR-Cas9 以用作E. histolytica的遗传工具. 我们选择了包含引导 RNA (gRNA) 和 Cas9 核酸酶表达盒的单一质粒方法。阿米巴 U6 启动子用于驱动 gRNA 的表达,并通过 Northern 印迹分析确认其表达。使用与 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的转染细胞系。使用该系统,我们能够在用小分子配体甲氧苄啶 (TMP) 处理后 16 小时诱导 ddCas9 表达。表达 ddCas9 和 Luc-gRNA 或非特异性 (NS)-gRNA 的稳定细胞系用含有 Luc-gRNA 靶向的突变荧光素酶基因 (pDeadLuc) 的质粒和另一个具有截短荧光素酶基因 (pDonorLuc) 的质粒瞬时转染以恢复荧光素酶的表达和随后的活性。我们观察到表达 Luc-gRNA 的细胞系的发光信号增加,表明 Cas9 活性促进了同源重组。通过 PCR 检测到的嵌合 DNA 的存在支持了这一证据,并通过对通过同源重组获得的修复 DNA 进行测序来证实。我们相信这代表了 CRISPR/Cas9 系统在Entamoeba并提供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基因研究。

更新日期:2020-11-29
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