Cas9
清脆的
合成生物学
雅罗维亚
代谢工程
生物
反式激活crRNA
基因组工程
CRISPR干扰
RNA干扰
基因
计算生物学
基因组编辑
遗传学
核糖核酸
作者
Joshua Misa,Cory Schwartz
出处
期刊:Methods in molecular biology
日期:2021-01-01
卷期号:: 95-109
被引量:3
标识
DOI:10.1007/978-1-0716-1414-3_6
摘要
Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.
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