Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica
Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica
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DOI：
10.1021/acssynbio.9b00498
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发表时间：
2020-04-17
影响因子：
4.7
通讯作者：
Wheeldon, Ian
中科院分区：
文献类型：
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作者：
Ramesh, Adithya;Ong, Thomas;Wheeldon, Ian
解脂耶氏酵母因其能够将脂质积累到高水平而迅速成为一种具有生物技术重要意义的酵母。虽然存在一套合成生物学工具用于这种酵母的基因工程，但需要用于快速菌株生成的多用途工具。在这里，我们描述了一种双重目的的CRISPR-Cpf 1系统，它能够同时进行基因破坏和基因调控。将引导RNA间隔区长度截短至16 nt抑制核酸酶活性，但不与靶基因座结合，从而使Cpf 1融合的转录调控因子能够激活和抑制基因。使用Cpf 1-Mxi 1融合物证明了基因抑制，实现了mRNA的7倍减少，而使用Cpf 1-VPR的CRISPR激活使hrGFP表达增加了10倍。用长度为23-25 bp的gRNA实现了高效破坏，并且用截短的和全长gRNA的多重表达维持了效率和抑制水平。开发的CRISPR-Cpf 1系统应该在代谢工程、全基因组筛选和功能基因组学研究中证明是有用的。
Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1 -fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.