Department of Genetics and Molecular Biology, Centro de Investigaciones y Estudios Avanzados (CINVESTAV), Ave. IPN #2508, San Pedro Zacatenco, Gustavo A. Madero, 07360 Mexico City, Mexico.
Department of Genetics and Molecular Biology, Centro de Investigaciones y Estudios Avanzados (CINVESTAV), Ave. IPN #2508, San Pedro Zacatenco, Gustavo A. Madero, 07360 Mexico City, Mexico.
Plasmid. 2022 Jul;122:102641. doi: 10.1016/j.plasmid.2022.102641. Epub 2022 Aug 8.
Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.
十二指肠贾第鞭毛虫是一种双核的微需氧原生动物，可引起贾第虫病。迄今为止，已经采用了几种分子方法来了解该寄生虫原生动物中各种细胞过程的分子机制。然而，许多参与这些过程的基因的作用需要进一步分析。CRISPR 干扰（CRISPRi）系统已被广泛用于包括贾第虫在内的几种寄生虫中用于基因沉默的组成型表达系统。这项工作的目的是在贾第虫中实施一种可调谐的 t-CRISPRi 系统，以沉默丰富，中等和低表达的基因，通过构建用于表达 gRNA 和 dCas9 的优化和诱导性质粒来实现。使用强力霉素诱导的 pRan 启动子表达 dCas9 和每个 gRNA，通过转染的滋养体中的 Western blot 和免疫荧光来始终如一地确认 dCas9 的表达和核定位。对α-微管蛋白（高表达），贾第蛋白酶 1（中度表达）和 Sir2 和 Sir4（低表达）基因进行转录抑制。通过 dCas9 强力霉素诱导引起的α-微管蛋白基因敲低导致其蛋白表达降低了 50％和 60％，分别在 24 和 48 小时。这导致鞭毛形态发生改变。giardipain-1 的敲低导致蛋白表达降低了 40％和 50％，分别在 12 和 24 小时，而不影响滋养体活力，与这一致的是，giardipain-1 敲低的同工酶分析显示 giardipain-1 蛋白酶活性降低。当抑制 sirtuins 表达时，尽管获得了完全抑制，但滋养体活力受到损害。这种方法为产生特定基因敲低的定制抑制提供了分子工具。
