Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis
Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis
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DOI：
10.3390/v10120732
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发表时间：
2018-12-01
期刊：
影响因子：
4.7
通讯作者：
Mahfouz, Magdy
中科院分区：
文献类型：
--
作者：
Aman, Rashid;Mahas, Ahmed;Mahfouz, Magdy
规则间隔短回文重复序列（CRISPR）和CRISPR相关（Cas）系统是帮助原核生物抵御RNA和DNA病毒的关键免疫机制。CRISPR/Cas9在基础研究和生物技术中具有广泛的应用，并已广泛用于真核生物的基因组工程和基因功能分析。最近开发的CRISPR/Cas 13系统靶向RNA而不是DNA，因此为转录组工程和对抗RNA病毒提供了新的潜力。在这里，我们使用CRISPR/LshCas 13 a稳定地工程化拟南芥，用于干扰芜菁花叶病毒（TuMV）的RNA基因组。我们的数据表明，与靶向TuMV RNA基因组其他区域的crRNA相比，将Cas 13 a引导至编码辅助组分蛋白酶沉默抑制子（HC-Pro）或GFP靶标2（GFP-T2）的序列的CRISPR RNA（crRNA）提供了更好的干扰。这项工作证明了CRISPR/Cas 13作为一种抗病毒策略来阻止RNA病毒的令人兴奋的潜力，并鼓励寻找更强大和有效的Cas 13变体或可以靶向RNA的CRISPR系统。
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.