利用CRISPR/Cas9系统改造酿酒酵母的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2022-1534

摘要/Abstract

摘要：

酿酒酵母是常用的工业生产菌株，改造酿酒酵母以提高其生产性能极为重要。然而，传统的改造方法存在步骤繁琐、周期长等问题。CRISPR/Cas9系统是在细菌和古细菌发现的自适应防御系统。目前CRISPR/Cas9系统已经成为基因组编辑的有力工具，能够同时改造酿酒酵母的多个基因。本文综述CRISPR/Cas9系统各组分的作用和系统的构建，介绍sgRNA靶序列的设计原则和代表性设计工具，总结对酿酒酵母进行多基因编辑的策略，以及CRISPR/Cas9系统存在脱靶和编辑效率低的问题和应对展望，为更好地应用CRISPR/Cas9系统改造酿酒酵母提供参考。

关键词: CRISPR/Cas9系统的构建, 多基因编辑, 靶序列的设计, 脱靶

Abstract:

Saccharomyces cerevisiae is one of widely used industrial strains, it is extremely important to increase its performance via modifying S. cerevisiae; however, there are cumbersome steps and long-cycle by traditional modifying method. CRISPR/Cas9 system is an adaptive defense system found in bacteria and archaea. At present, the CRISPR/Cas9 system has become a powerful tool for genome editing, which can simultaneously modify multiple genes of S. cerevisiae. This review provided a brief overview of the function and construction of CRISPR/Cas9 system in S. cerevisiae gene editing, introduced the design rules and representative design tools of sgRNA targeting sequences, as well as summarized the multiple-gene editing strategy. Moreover, the review discussed the challenges in application of CRISPR/Cas9 system, including off target and low editing efficiency, and their control measures, which may provide the references for better applying CRISPR/Cas9 system modifying S. cerevisiae.

Key words: construction of CRISPR/Cas9 system, multiplex gene editing, design of target sequence, off-target

陈小玲, 廖东庆, 黄尚飞, 陈英, 芦志龙, 陈东. 利用CRISPR/Cas9系统改造酿酒酵母的研究进展[J]. 生物技术通报, 2023, 39(8): 148-158.

CHEN Xiao-ling, LIAO Dong-qing, HUANG Shang-fei, CHEN Ying, LU Zhi-long, CHEN Dong. Advances in CRISPR/Cas9 System Modifying Saccharomycescerevisiae[J]. Biotechnology Bulletin, 2023, 39(8): 148-158.

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名称 Name	链接 Link	物种Species	优点 Advantage	缺点 Disadvantage	参考文献Reference
GuideScan	-	任何微生物	能以目标酿酒酵母的基因组为参考基因组、有助于减少脱靶位点，设计的靶序列比the mit.edu web interface^[30]、CRISPRscan^[42]及E-CRISP^[56]的好	需在本地计算机安装，需使用命令行，需提供参考基因组	[45]
Yeastriction	http://yeastriction.tnw.tudelft.nl/	酿酒酵母	设计的靶序列比ChopChop^[57]的好，提供33株酿酒酵母的基因组作为备选的参考基因组，不需使用命令行	容易脱靶（除非参考基因组是目标酿酒酵母的基因组）	[26]
CRISPy	http://staff.biosustain.dtu.dk/laeb/crispy_yeast/	酿酒酵母	不需上传参考基因组	只能以酿酒酵母S288C的基因组为参考基因组，为其他酿酒酵母设计靶序列时，容易脱靶	[28]
CRISPy-web	http://crispy.secondarymetabolites.org/	任何微生物	免安装，不需使用命令行，能以目标酿酒酵母的基因组为参考基因组、有助于减少脱靶位点	需上传参考基因组	[55]

名称 Name	链接 Link	物种Species	优点 Advantage	缺点 Disadvantage	参考文献Reference
GuideScan	-	任何微生物	能以目标酿酒酵母的基因组为参考基因组、有助于减少脱靶位点，设计的靶序列比the mit.edu web interface^[30]、CRISPRscan^[42]及E-CRISP^[56]的好	需在本地计算机安装，需使用命令行，需提供参考基因组	[45]
Yeastriction	http://yeastriction.tnw.tudelft.nl/	酿酒酵母	设计的靶序列比ChopChop^[57]的好，提供33株酿酒酵母的基因组作为备选的参考基因组，不需使用命令行	容易脱靶（除非参考基因组是目标酿酒酵母的基因组）	[26]
CRISPy	http://staff.biosustain.dtu.dk/laeb/crispy_yeast/	酿酒酵母	不需上传参考基因组	只能以酿酒酵母S288C的基因组为参考基因组，为其他酿酒酵母设计靶序列时，容易脱靶	[28]
CRISPy-web	http://crispy.secondarymetabolites.org/	任何微生物	免安装，不需使用命令行，能以目标酿酒酵母的基因组为参考基因组、有助于减少脱靶位点	需上传参考基因组	[55]