CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用

doi:10.12211/2096-8280.2022-076

合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 738-755.DOI: 10.12211/2096-8280.2022-076

CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用

林继聪¹, 邹根², 刘宏民¹, 魏勇军¹

^1.郑州大学药学院，郑州大学药物研究院，郑州大学合成生物学实验室，药物关键制备技术教育部重点实验室，河南郑州 450001
^2.上海市农业科学院食用菌研究所，农业农村部南方食用菌资源利用重点实验室，国家食用菌工程技术研究中心，上海 201403

收稿日期:2022-12-28 修回日期:2023-02-27 出版日期:2023-08-31 发布日期:2023-09-14
通讯作者: 刘宏民,魏勇军
作者简介:林继聪（1999—），男，硕士研究生。研究方向为丝状真菌合成生物学。E-mail：ljc990101@163.com
刘宏民（1960—），男，博士，教授。研究方向为药物设计与合成。E-mail：liuhm@zzu.edu.cn
魏勇军（1986—），男，博士，副教授。研究方向为合成微生物组学。E-mail：yongjunwei@zzu.edu.cn
基金资助:
国家自然科学基金(32111530179)

Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi

Jicong LIN¹, Gen ZOU², Hongmin LIU¹, Yongjun WEI¹

^1.School of Pharmaceutical Sciences，Institute of Drug Discovery and Development，Laboratory of Synthetic Biology，Key Laboratory of Advanced Drug Preparation Technologies，Ministry of Education of China，Zhengzhou University，Zhengzhou 450001，Henan，China
^2.Institute of Edible Fungi，Shanghai Academy of Agricultural Sciences，Southern Key Laboratory of Edible Fungus Resource Utilization，Ministry of Agriculture，National Engineering Research Center of Edible Fungi，Shanghai 201403，China

Received:2022-12-28 Revised:2023-02-27 Online:2023-08-31 Published:2023-09-14
Contact: Hongmin LIU, Yongjun WEI

摘要/Abstract

摘要：

丝状真菌能够合成抗生素、色素、酶制剂、激素等多种天然产物，广泛应用于医药、化工、农业和基础生物学研究等领域。丝状真菌遗传背景复杂，阻碍了对其的进一步开发利用。基因组编辑是基于核酸酶对基因组位点特异性序列进行靶向切割，产生双链断裂，从而通过非同源末端连接或同源重组进行修复的技术。其中，CRISPR（clustered regularly interspaced short palindromic repeats）系统是目前使用最普遍的基因组编辑技术，已在丝状真菌遗传育种、基因改造和多种天然产物合成等方面进行了大量应用。本文总结了丝状真菌CRISPR/Cas的技术原理、元件表达、递送方式及该系统在次级代谢产物等研究中的应用。对于脱靶效应以及转化率低的问题，本文讨论了可能的解决方法。在此基础上，展望了基于CRISPR/Cas的基因组编辑技术在真菌基因功能表征、天然产物合成代谢途径解析与重构、高效丝状真菌底盘细胞构建、天然产物合成等方面的应用。

关键词: 丝状真菌, CRISPR/Cas系统, 次级代谢产物, 基因敲除, 异源表达

Abstract:

Filamentous fungi are the producers of antibiotics, pigments, enzymes, hormones, and other natural products, which are widely applied in the industries of medicine, chemical engineering, agriculture, and basic biology studies. The genetic background of filamentous fungi is complex, and molecular biology studies of filamentous fungi are difficult. Genome editing can cut specific sites of the genomic double-stranded DNA, to finish the insertion, deletion, or replacement of genomic information in vivo based on non-homologous end joining repair or homologous recombination repair. CRISPR system is the most widely used genome editing technology, which has been applied in genetic breeding, metabolic engineering, and the production of valuable natural products with filamentous fungi. The secondary metabolites of filamentous fungi, the gene editing principles, biopart design and expression, and delivery strategy of the CRISPR/Cas system were introduced, and the application of CRISPR/Cas system of filamentous fungi was summarized. Possible solutions to solve the problems of off-target effect and low conversion rates of gene editing were discussed. Besides, the application of the CRISPR/Cas system in the characterization of fungal gene function, natural product biosynthetic pathway recovery and rebuilding, construction of efficient filamentous fungi chassis cells, and natural product biosynthesis were also discussed. {L-End}

Key words: filamentous fungi, CRISPR-Cas system, secondary metabolites, gene editing, heterologous expression

中图分类号:

Q812

林继聪, 邹根, 刘宏民, 魏勇军. CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用[J]. 合成生物学, 2023, 4(4): 738-755.

Jicong LIN, Gen ZOU, Hongmin LIU, Yongjun WEI. Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi[J]. Synthetic Biology Journal, 2023, 4(4): 738-755.

图/表 4

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丝状真菌	sgRNA 启动子	表达方式	靶标基因	编辑效率	参考文献
里氏木霉		体内表达密码子优化的Cas9和体外转录的sgRNA	ura5, lae1, vib1, clr2	单基因93％～100％；多基因4.2％～45％	[38]
里氏木霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA1的表达	ura5	8%～10%	[104]
里氏木霉	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	lae1	36.67%	[105]
里氏木霉		体外组装的核糖核蛋白（RNP）复合物	ura5, lae1, cbh1, cbh2, eg1	56.52%～100%	[73]
草酸青霉	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	bgl2, creA	30%～80％	[105]
鲁本斯青霉	U6, tRNA, utp25	体内表达密码子优化的Cas9与U6，tRNA和utp25启动子驱动sgRNA的表达；体外组装的核糖核蛋白（RNP）复合物	pks17, roqA, lovF, hcpA, pcbA, penDE, chyA	60%～100%	[57]
黑曲霉	tRNA	体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达	albA, olvA, glaA	20％～93％	[61]
黑曲霉	5S rRNA	体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达	albA, fum5, fum1	33％～100％	[62]
米曲霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达	wA, pyrG, yA	10%～100％	[106]
米曲霉	U6	携带Cas9和sgRNA的自主复制质粒	wA, pyrG, yA	55.6％～100％	[68]
烟曲霉		体内表达密码子优化的Cas9和体外转录的sgRNA	pksP, cnaA	95%～100%	[107]
藤仓镰刀菌	P_gpdA, U6, 5S rRNA	体内表达密码子优化的Cas9与P_gpdA 、U6、5S rRNA启动子驱动sgRNA的表达	fcc1, ura3, ppt1	37.5％～79.2％	[49]
Glarea lozoyensis	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	gloA, gloF	28%	[93]
嗜热毁丝霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达	cre-1, res-1, gh1-1, alp-1	单基因95％；多基因22%～70%	[59]
嗜热毁丝霉	U6	体内表达密码子优化的Cas12a和U6启动子驱动sgRNA的表达	cre-1, res-1, gh1-1	单基因90％；多基因13％～41％	[108]
稻瘟病菌		体外组装的Cas12a核糖核蛋白（RNP）复合物	BUF1, FKBP12, FTR1, BAS4, AVRPI9	25％～77%	[109]

丝状真菌	sgRNA 启动子	表达方式	靶标基因	编辑效率	参考文献
里氏木霉		体内表达密码子优化的Cas9和体外转录的sgRNA	ura5, lae1, vib1, clr2	单基因93％～100％；多基因4.2％～45％	[38]
里氏木霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA1的表达	ura5	8%～10%	[104]
里氏木霉	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	lae1	36.67%	[105]
里氏木霉		体外组装的核糖核蛋白（RNP）复合物	ura5, lae1, cbh1, cbh2, eg1	56.52%～100%	[73]
草酸青霉	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	bgl2, creA	30%～80％	[105]
鲁本斯青霉	U6, tRNA, utp25	体内表达密码子优化的Cas9与U6，tRNA和utp25启动子驱动sgRNA的表达；体外组装的核糖核蛋白（RNP）复合物	pks17, roqA, lovF, hcpA, pcbA, penDE, chyA	60%～100%	[57]
黑曲霉	tRNA	体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达	albA, olvA, glaA	20％～93％	[61]
黑曲霉	5S rRNA	体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达	albA, fum5, fum1	33％～100％	[62]
米曲霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达	wA, pyrG, yA	10%～100％	[106]
米曲霉	U6	携带Cas9和sgRNA的自主复制质粒	wA, pyrG, yA	55.6％～100％	[68]
烟曲霉		体内表达密码子优化的Cas9和体外转录的sgRNA	pksP, cnaA	95%～100%	[107]
藤仓镰刀菌	P_gpdA, U6, 5S rRNA	体内表达密码子优化的Cas9与P_gpdA 、U6、5S rRNA启动子驱动sgRNA的表达	fcc1, ura3, ppt1	37.5％～79.2％	[49]
Glarea lozoyensis	5S rRNA	体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达	gloA, gloF	28%	[93]
嗜热毁丝霉	U6	体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达	cre-1, res-1, gh1-1, alp-1	单基因95％；多基因22%～70%	[59]
嗜热毁丝霉	U6	体内表达密码子优化的Cas12a和U6启动子驱动sgRNA的表达	cre-1, res-1, gh1-1	单基因90％；多基因13％～41％	[108]
稻瘟病菌		体外组装的Cas12a核糖核蛋白（RNP）复合物	BUF1, FKBP12, FTR1, BAS4, AVRPI9	25％～77%	[109]

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CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用

Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi

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