Robust and stable transcriptional repression in Giardia using CRISPRi.
Robust and stable transcriptional repression in Giardia using CRISPRi.
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DOI：
10.1091/mbc.e18-09-0605
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发表时间：
2019-01-01
影响因子：
3.3
通讯作者：
Dawson SC
中科院分区：
文献类型：
--
作者：
McInally SG;Hagen KD;Nosala C;Williams J;Nguyen K;Booker J;Jones K;Dawson SC
蓝氏贾第鞭毛虫是一种双核原生生物寄生虫，在世界范围内引起严重的疟疾。由于无法将Cas9靶向两个细胞核，加上缺乏非同源末端连接和阳性选择标记，使得CRISPR/Cas9介导的遗传工具无法适应这种广泛分布的寄生虫。CRISPR干扰（CRISPRi）是CRISPR/Cas9系统的修饰，其将催化失活的Cas9（dCas 9）引导至靶基因座以进行稳定的转录抑制。使用贾第虫核定位信号将dCas 9靶向两个核，我们开发了贾第虫中外源和内源基因的有效且稳定的CRISPRi介导的转录抑制。具体地，驱动蛋白-2a和13的CRISPRi敲低导致严重的鞭毛长度缺陷，其反映了吗啉代敲低的缺陷。敲低腹盘MBP蛋白也会导致严重的结构缺陷，这是非常普遍的，并坚持在人口中超过5天以上的时间比缺陷与短暂的吗啉基敲低。通过串联表达两个指导RNA以同时敲低驱动蛋白-13和MBP，我们创建了具有鞭毛长度和圆盘缺陷的稳定的双敲低菌株。CRISPRi在多倍体贾第虫中的效率和简单性允许快速评估敲除表型，并突出了CRISPRi在新兴模型系统中的实用性。
Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.