Design Strategies of sgRNA and Its Expression and Application in Filamentous Fungi

doi:10.13560/j.cnki.biotech.bull.1985.2025-1268

Abstract

Abstract:

Filamentous fungi possess unique morphological and genetic features that hinder large-scale and precise genome modifications using conventional genetic tools. Recently, the CRISPR-Cas9 system has become a powerful approach for fungal genome engineering owing to its structural simplicity, operational flexibility, and cost-effectiveness. As the key determinant of targeting efficiency and specificity, single-guide RNA (sgRNA) plays a central role in the performance of CRISPR-Cas9 and its derivatives. This review summarizes current strategies for sgRNA design, construction, and expression in filamentous fungi, and outlines its applications in functional gene analysis, metabolic engineering, and industrial strain development. Despite its great potential, the review points out that CRISPR-Cas9 editing in filamentous fungi often suffers from low efficiency and off-target effects, primarily due to suboptimal sgRNA design, poor transcriptional activity, and host-dependent DNA repair mechanisms. Finally, the review discusses the optimization approaches and future applications of sgRNA, aiming to facilitate the establishment of efficient and reliable sgRNA-based gene editing platforms for diverse filamentous fungal species.

Key words: sgRNA design, filamentous fungi, gene editing, expression strategies

YANG Jia-ni, WU Tao, ZHANG Hong-yang, HUANG Bing-guang, LU Min, HE Jia-jie, RUAN Hai-hua. Design Strategies of sgRNA and Its Expression and Application in Filamentous Fungi[J]. Biotechnology Bulletin, 2026, 42(9): 262-275.

Figures/Tables 8

References 93

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sgRNA启动子类型 Type of sgRNA promoter		表达方式 Expression method	转化方式 Conversion method	核定位信号 NLS	修复方式 DSB repair	效率 Efficiency （%）	应用 Application
RNA聚合酶Ⅱ型启动子Pol Ⅱ promoter	Pgpda	Plasmid	PMT	stuA	NHEJ	64.2	Alternaria alternata^［19］
	Pgpda	Plasmid	PMT	SV40	NHEJ	/	Penicillium rubens^［20］
	Ptrpc	Plasmid	AMT	c-Myc	NHEJ /HDR	18-26	Cordyceps militaris^［21］
RNA聚合酶Ⅲ型启动子Pol III promoter	U6	Plasmid	PMT	H2B	HDR	80-100	Fusarium^［22］
		Plasmid	PMT	SV40	BE	47.36-100	Aspergillus niger^［23］
		Plasmid	PMT	SV40	MMEJ	67	Aspergillus fumigatus^［24］
	U3	Plasmid	PMT	SV40	HDR	23.55-66.7	Aspergillus niger^［25］
	5SrRNA	Plasmid	PMT	SV40	HDR	36.67	Penicillium oxalicum^［26］
	5SrRNA	Plasmid	PMT	HTB/VEL	HDR	58.3-79.2	Fusarium fujikuroi^［27］
	tRNA	Plasmid	PMT	SV40	HDR	45-80	Aspergillus aculeatus^［28］
	SNR52	Plasmid	PMT	SV40	HDR	25-53	Aspergillus fumigatus^［29］
体外 In vitro	T7	Plasmid and vitro	PMT	SV40	MMEJ	100	Aspergillus nidulans^［30］
体外 In vitro	/	RNP	PMT	SV40	NHEJ	68	Botrytis cinerea^［31］

sgRNA启动子类型 Type of sgRNA promoter		表达方式 Expression method	转化方式 Conversion method	核定位信号 NLS	修复方式 DSB repair	效率 Efficiency （%）	应用 Application
RNA聚合酶Ⅱ型启动子Pol Ⅱ promoter	Pgpda	Plasmid	PMT	stuA	NHEJ	64.2	Alternaria alternata^［19］
	Pgpda	Plasmid	PMT	SV40	NHEJ	/	Penicillium rubens^［20］
	Ptrpc	Plasmid	AMT	c-Myc	NHEJ /HDR	18-26	Cordyceps militaris^［21］
RNA聚合酶Ⅲ型启动子Pol III promoter	U6	Plasmid	PMT	H2B	HDR	80-100	Fusarium^［22］
		Plasmid	PMT	SV40	BE	47.36-100	Aspergillus niger^［23］
		Plasmid	PMT	SV40	MMEJ	67	Aspergillus fumigatus^［24］
	U3	Plasmid	PMT	SV40	HDR	23.55-66.7	Aspergillus niger^［25］
	5SrRNA	Plasmid	PMT	SV40	HDR	36.67	Penicillium oxalicum^［26］
	5SrRNA	Plasmid	PMT	HTB/VEL	HDR	58.3-79.2	Fusarium fujikuroi^［27］
	tRNA	Plasmid	PMT	SV40	HDR	45-80	Aspergillus aculeatus^［28］
	SNR52	Plasmid	PMT	SV40	HDR	25-53	Aspergillus fumigatus^［29］
体外 In vitro	T7	Plasmid and vitro	PMT	SV40	MMEJ	100	Aspergillus nidulans^［30］
体外 In vitro	/	RNP	PMT	SV40	NHEJ	68	Botrytis cinerea^［31］

物种 Species	启动子类型 Promoter type	递送方式 Delivery method	编辑效率 Editing efficiency
Myceliophthora thermophila^［71］	U6 promoter	sgRNA表达盒	在多重编辑中，单个基因的编辑效率范围在13%-41%之间
Aspergillus niger^［55］	tRNA promoter sgRNA array	pLM2质粒递送	三重整合效率达到23.5%
Pleurotus ostreatus^［72］	tRNA promoter sgRNA array	质粒递送	经过启动子和tRNA序列优化后，效率提升至20%
Trichoderma reesei^［73］	5SrRNA promoter tRNA-sgRNA array	质粒递送	成功实现了cbh1和cbh2的双基因敲除
Aspergillus niger^［74］	T7 promoter	RNP递送	采用双sgRNA策略构建pyrG营养缺陷型突变体，阳性率达到100%

物种 Species	启动子类型 Promoter type	递送方式 Delivery method	编辑效率 Editing efficiency
Myceliophthora thermophila^［71］	U6 promoter	sgRNA表达盒	在多重编辑中，单个基因的编辑效率范围在13%-41%之间
Aspergillus niger^［55］	tRNA promoter sgRNA array	pLM2质粒递送	三重整合效率达到23.5%
Pleurotus ostreatus^［72］	tRNA promoter sgRNA array	质粒递送	经过启动子和tRNA序列优化后，效率提升至20%
Trichoderma reesei^［73］	5SrRNA promoter tRNA-sgRNA array	质粒递送	成功实现了cbh1和cbh2的双基因敲除
Aspergillus niger^［74］	T7 promoter	RNP递送	采用双sgRNA策略构建pyrG营养缺陷型突变体，阳性率达到100%