Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae,ACS Synthetic Biology

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Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-12-01 00:00:00 , DOI: 10.1021/acssynbio.7b00259
Raphael Ferreira _{1,

2} , Christos Skrekas ₁ , Jens Nielsen _{1,

2,

3} , Florian David _{1,

2}

Affiliation

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.

中文翻译：

在酿酒酵母中使用 Csy4 进行多重 CRISPR/Cas9 基因组编辑和基因调控

成簇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的发展。然而，在酿酒酵母中开发强大的多重分析工具方面还没有做出足够的努力。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 处理能力，从单个转录本生成多个 gRNA，用于酿酒酵母中的基因组编辑和基因干扰应用。在基因组编辑方面，我们对FAA1 、 FAA4 、 POX1和TES1进行了四重删除，在测试的 24 个菌落中达到了 96% 的效率。然后，我们利用该系统有效地转录调控OLE1 、 HMG1和ACS1这三个基因。因此，我们证明可以使用 Csy4 以快速有效的方式进行多重基因组编辑和基因调控。

更新日期：2017-12-01

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