丝状真菌(filamentous fungi)具有独特的形态和细胞构造,与人类健康和工农业生产息息相关,对这类生物资源的开发和利用高度依赖高效的基因编辑平台.然而,由于丝状真菌复杂多样的遗传背景,使用传统的基因编辑技术较难实现大范围的基因编辑,极大地妨碍了丝状真菌的遗传学研究.CRISPR/Cas(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein)技术的出现,打破了这一困境,促进了不同种属和不同来源的丝状真菌的基因编辑,为丝状真菌的基础和应用研究带来了革命性的突破.本文简述了 CRISPR/Cas系统的作用机理、分类及基于CRISPR的各种新型技术,归纳总结了丝状真菌中现有的CRISPR/Cas9系统功能组分、多种新兴CRISPR/Cas技术在丝状真菌中的应用现状以及海洋真菌中的CRISPR/Cas技术的应用情况.最后,对CRISPR/Cas系统在丝状真菌中应用进展缓慢、编辑效率低和脱靶效应等问题以及针对这些问题的潜在解决方法进行总结和展望,以期为不同类型的丝状真菌基因编辑平台的构建提供参考.
CRISPR/Cas systems and their applications in gene editing with filamentous fungi
Filamentous fungi,which present distinct morphology and cell structure,play a critical role in human health as well as industrial and agricultural production.However,the unique characteristics of filamentous fungi make them difficult to be manipulated with traditional genetic engineering methods.Thus,the development of an efficient gene editing system is essential for exploring biological resources and understanding metabolic processes in filamentous fungi.The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein(CRISPR/Cas)system promotes more efficient and effective gene editing in different species,and brings a revolutionary breakthrough in fungal fundamental research and applications.In this review,we first briefly introduce the history,working mechanism,and classifications of the CRISPR/Cas mediated gene editing system.Next,we comment the functional components of CRISPR/Cas9 such as selective marker,Cas9 and gRNA and the delivery methods of these components in various filamentous fungi.Furthermore,we systematically discuss the applications of CRISPR related technologies,including CR1SPR/Casl2,base-editor,CRISPRa,CRISPRi and CRISPR mediated epigenetic regulation,in the genetic engineering of filamentous fungi,particularly in marine-derived filamentous fungi.Finally,we address challenges with relative low gene editing efficiency and off-targets effects in engineering filamentous fungi,and highlight the potential solutions for developing novel CRISPR/Cas-based gene editing systems.This review can provide guidance for developing an efficient gene editing platform in filamentous fungi and pave the way for further exploration of the secondary metabolites and establishment of robust fungal cell factories.
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