sgRNA的设计策略及其在丝状真菌中的表达与应用

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

摘要/Abstract

摘要：

丝状真菌因其独特的结构和复杂的遗传背景，传统的基因编辑技术无法实现对其大范围的基因改造。近年来，CRISPR-Cas9系统因结构简单、操作便捷和成本低廉等优势，逐渐成为丝状真菌基因工程研究的重要工具，而sgRNA是CRISPR-Cas9基因编辑系统及其衍生技术中的重要元件。本文归纳了sgRNA的设计原则和构建方法及其在丝状真菌中的转化与表达策略，分析了sgRNA在功能基因解析、菌株代谢工程改造和工业菌株放大生产中的应用现状，同时指出丝状真菌CRISPR-Cas9基因编辑过程中普遍存在的编辑效率低和脱靶效应等问题，主要与sgRNA的设计合理性、表达效率及宿主修复机制密切相关。最后，针对sgRNA的优化方向与未来应用进行展望，以期为构建高效、稳定的丝状真菌基因编辑平台提供参考。

关键词: sgRNA的设计, 丝状真菌, 基因编辑, 表达策略

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

杨佳妮, 武陶, 张宏扬, 黄冰光, 卢敏, 何佳杰, 阮海华. sgRNA的设计策略及其在丝状真菌中的表达与应用[J]. 生物技术通报, 2026, 42(9): 262-275.

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.

图/表 8

图1 sgRNA结构示意图

Fig. 1 Schematic diagram of sgRNA structure

表1 sgRNA在CRISPR-Cas9系统中的应用

Table 1 Applications of sgRNA in the CRISPR-Cas9 system

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］

图2 sgRNA表达方式示意图A：体外转录，RNP复合体直接进入细胞核执行基因编辑功能。B：体内表达，Cas9蛋白和sgRNA序列组装在质粒上。质粒进入细胞核后，Cas9序列转录成mRNA，然后转运到细胞质中翻译成Cas9蛋白，进入细胞核与sgRNA协作执行编辑功能

Fig. 2 Schematic representation of sgRNA expression patternsA: In vitro transcription, where the RNP complex directly enters the nucleus to perform gene editing functions. B: In vivo expression, where the Cas9 protein and sgRNA sequences are assembled on a plasmid. After the plasmid enters the nucleus, the Cas9 sequence is transcribed into mRNA, which is then transported to the cytoplasm for translation into the Cas9 protein. This protein subsequently enters the nucleus to collaborate with the sgRNA and execute editing functions

表2 三种代表性RNA聚合酶Ⅲ型启动子的比较

Table 2 Comparison of three representative RNA Polymerase III promoters

Pol III promoter

优点

Merit

局限性

Limitation

应用

Application

转录效率高；

具有明确的终止信号^［46］

物种特异性强；sgRNA需要以5′-G开头，限制了序列的选择

Aspergillus oryzae^［49］

Humicola insolens^［50］

Aspergillus fumigatus^［51］

Trichoderma reesei^［52］

5S rRNA

转录水平高且稳定；转录起始位点精确；转录终止信号简单（4-6个连续的T）^［47］

具有物种特异性，需要进行验证和优化；不直接适用于多重sgRNA表达

tRNA

高度的基因组拷贝数能够促进高效转录；能够形成多顺反子的“tRNA-sgRNA”阵列，实现多重表达^［48］

复杂的阵列设计需要优化；取决于内源性tRNA的加工效率；转录起始位点可能存在变异

Aspergillus niger^［25］

图3 丝状真菌中常见的3种外源DNA的转化方式EP（电击转化）：通过高强度电场在细胞膜上产生暂时孔洞，使外源DNA进入细胞内；PMT（原生质体介导的转化）：酶解去除细胞壁后，利用PEG促进DNA与细胞膜融合，实现外源DNA的导入；AMT（农杆菌介导的转化）：利用根癌农杆菌天然的基因传递能力，Ti质粒上的转移DNA（T-DNA）在Vir基因的介导下被转运到植物细胞，并随机整合到目标基因组中，实现外源基因导入

Fig. 3 Three common methods for transforming exogenous DNA into filamentous fungiEP (Electroporation): DNA introduced via electrical perforation. PMT (Protoplast-mediated transformation): Exogenous DNA absorbed by protoplasts after cell wall removal via enzymatic digestion, facilitated by PEG. AMT (Agrobacterium-mediated transformation): Utilizes the natural gene transfer capability of rhizobium-associated Agrobacterium; the Ti plasmid in Agrobacterium contains a specific DNA segment (T-DNA) that, upon activation of the Vir gene, transfers and integrates T-DNA into the targeted genome

图4 sgRNA在丝状真菌中的应用

Fig. 4 Application of sgRNA in filamentous fungi

表3 丝状真菌多位点编辑中sgRNA表达方式及其编辑效率

Table 3 The sgRNA expression strategy and editing efficiency in multi-site editing of filamentous fungi

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%

图5 sgRNA的未来应用与展望

Fig. 5 Future applications and prospects of sgRNA

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