Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae
Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae
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DOI：
10.1021/acssynbio.7b00259
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发表时间：
2018-01-01
影响因子：
4.7
通讯作者：
David, Florian
中科院分区：
文献类型：
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作者：
Ferreira, Raphael;Skrekas, Christos;David, Florian
规则间隔短回文重复序列（CRISPR）技术极大地加速了菌株工程领域。然而，在酿酒酵母中开发稳健的多路复用工具的努力还不够。在这里，我们利用来自铜绿假单胞菌的细菌核糖核酸内切酶Csy4的RNA加工能力，从单个转录物产生多个gRNA，用于S.啤酒。在基因组编辑方面，我们对FAA1、FAA4、POX1和TES1进行了四重缺失，在测试的24个菌落中达到了96%的效率。然后，我们使用该系统有效地转录调控三个基因，OLE1，HMG1和ACS1。因此，我们证明了多重基因组编辑和基因调控可以使用Csy4以快速有效的方式进行。
Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the three genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4.