Ma Bin, Li Yimiao, Wang Tinghui, Li Dongming, Jia Shuang
School of Life Sciences, Inner Mongolia University, Hohhot 010070, China.
J Fungi (Basel). 2025 May 1;11(5):350. doi: 10.3390/jof11050350.
As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.
作为一类重要的微生物，丝状真菌在蛋白质分泌、次级代谢产物合成及环境污染控制中发挥着关键作用。然而，顶端生长、异核体、低同源重组（HR）效率以及遗传标记稀缺等特性意味着传统基因编辑技术在丝状真菌中的应用面临巨大挑战。近年来，RNA介导的CRISPR/Cas（成簇规律间隔短回文重复序列/CRISPR相关蛋白）系统引入丝状真菌，彻底改变了丝状真菌的基因编辑方式。此外，持续表达的CRISPR系统显著提高了编辑效率，而优化的sgRNA设计和降低的cas9浓度有效降低了脱靶效应，进一步增强了该技术的安全性和可靠性。在本综述中，我们系统分析了CRISPR/Cas9的分子机制和调控因子，着重探讨其在丝状真菌中的表达系统优化及转化效率提升，并揭示HR和非同源末端连接（NHEJ）途径在基因编辑中的核心调控作用。基于对各种丝状真菌应用的分析，本综述揭示了CRISPR/Cas9在增强蛋白质分泌方面的突出优势，阐述了过去十年中次级代谢途径重建及污染物降解情况，为该技术的优化和工程应用提供了理论依据和实践指导。
