CRISPR/Cas9基因编辑系统在食药用真菌中的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2021-1055

摘要/Abstract

摘要：

CRISPR/Cas9是目前最成功的基因组精准编辑技术。迄今为止至少在9种食药用真菌中已有相关的应用报道,包括刺芹侧耳（杏鲍菇）、糙皮侧耳（平菇）、双孢蘑菇、灵芝、裂褶菌、灰盖鬼伞、金针菇、蛹虫草和竹黄菌。本文综述了CRISPR/Cas9系统的工作原理及递送策略,针对在食药用真菌中应用存在的一些问题进行分析,并提出优化方案。最后借鉴目前CRISPR技术发展的前沿方向,如多基因编辑、碱基编辑、引导编辑、转录调控等,对未来在食药用真菌领域的应用前景进行了展望。

关键词: CRISPR/Cas9, 食药用真菌, 基因组编辑, RNP

Abstract:

CRISPR/Cas9 is currently the most successful precise genome editing technology. So far,there have been relevant application reports in at least nine edible and medicinal fungi,including Pleurotus eryngii,Pleurotus ostreatus,Agaricus bisporus,Ganoderma lucidum,Schizophyllum commune,Coprinopsis cinerea,Flammulina velutipes,Cordyceps militaris and Shiraia bambusicola. This article briefly describes the mechanism and delivery strategies of the CRISPR/Cas9 system,and proposes solutions for the application in edible and medicinal fungi. Last but not least,referring to the current frontier development of CRISPR technology,such as multiplex gene editing, base editing, prime editing and transcription regulation, etc., we give certain enlightenment to the future application prospects in the field of edible and medicinal fungi.

Key words: CRISPR/Cas9, edible and medicinal fungi, genome editing, RNP

刘笑天, 仇昊, 田莉, 任昂, 赵明文. CRISPR/Cas9基因编辑系统在食药用真菌中的研究进展[J]. 生物技术通报, 2021, 37(11): 4-13.

LIU Xiao-tian, QIU Hao, TIAN Li, REN Ang, ZHAO Ming-wen. Research Progress in CRISPR/Cas9 Genome Editing System in Edible and Medicinal Fungi[J]. Biotechnology Bulletin, 2021, 37(11): 4-13.

图/表 2

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分类 Classifications	物种 Species	Cas9的表达 Expression of Cas9	gRNA的表达 Expression of gRNA	转化材料及递送方式 Materials and delivery methods	编辑模式 Editing methods	靶向基因和筛选标记 Target sites and marker	参考文献 References
担子菌 Basidiomycota	刺芹侧耳 P. eryngii	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	pyrG,cbx^r	2021^[9]
	糙皮侧耳 P. ostreatus	体外合成	体外合成	原生质体,PMT	NHEJ/HDR	pyrG,hyg^r	2021^[10]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	fcy1/pyrG,hyg^r	2021^[11]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	pcc1/clp1,hyg^r	2021^[12]
	双孢蘑菇 A. bisporus	内源gpd启动子表达Cas9	内源U6启动子	菌丝体,ATMT	NHEJ	ppo4,hyg^r	2020^[13]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3/GL17624,bar^r	2020^[14]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	ura3/cyp515018 /cyp505d13,cbx^r	2020^[15]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3,cbx^r	2017^[16]
	裂褶菌 S. commune	外源表达	体外转录	原生质体,PMT	HDR	hom2,nour^r	2019^[17]
	灰盖鬼伞 C. cinerea	内源CcDED1启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	gfp,hyg^r	2017^[18]
	金针菇 F. velutipes	内源gpd启动子表达密码子优化的Cas9	无	菌丝体,ATMT	未编辑	hyg^r	2017^[19]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源gpd启动子	菌丝体,ATMT	未编辑	Fvgpcr1/Fvgpcr2,hyg^r	2019^[20]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源H1启动子	原生质体,PMT	未编辑	hk1/hk2,hyg^r	2018^[21]
子囊菌 Ascomycota	蛹虫草 C. militaris	内源gpd启动子表达密码子优化的Cas9	T7体外转录	分生孢子,ATMT; 原生质体,PMT	NHEJ	ura3,blpR^r	2018^[22]
子囊菌 Ascomycota	竹黄菌 S. bambusicola	内源ef1α启动子表达Cas9	人内源U6启动子	原生质体,PMT	HDR	SbaPKS,hyg^r	2017^[23]

分类 Classifications	物种 Species	Cas9的表达 Expression of Cas9	gRNA的表达 Expression of gRNA	转化材料及递送方式 Materials and delivery methods	编辑模式 Editing methods	靶向基因和筛选标记 Target sites and marker	参考文献 References
担子菌 Basidiomycota	刺芹侧耳 P. eryngii	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	pyrG,cbx^r	2021^[9]
	糙皮侧耳 P. ostreatus	体外合成	体外合成	原生质体,PMT	NHEJ/HDR	pyrG,hyg^r	2021^[10]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	fcy1/pyrG,hyg^r	2021^[11]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	pcc1/clp1,hyg^r	2021^[12]
	双孢蘑菇 A. bisporus	内源gpd启动子表达Cas9	内源U6启动子	菌丝体,ATMT	NHEJ	ppo4,hyg^r	2020^[13]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3/GL17624,bar^r	2020^[14]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	ura3/cyp515018 /cyp505d13,cbx^r	2020^[15]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3,cbx^r	2017^[16]
	裂褶菌 S. commune	外源表达	体外转录	原生质体,PMT	HDR	hom2,nour^r	2019^[17]
	灰盖鬼伞 C. cinerea	内源CcDED1启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	gfp,hyg^r	2017^[18]
	金针菇 F. velutipes	内源gpd启动子表达密码子优化的Cas9	无	菌丝体,ATMT	未编辑	hyg^r	2017^[19]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源gpd启动子	菌丝体,ATMT	未编辑	Fvgpcr1/Fvgpcr2,hyg^r	2019^[20]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源H1启动子	原生质体,PMT	未编辑	hk1/hk2,hyg^r	2018^[21]
子囊菌 Ascomycota	蛹虫草 C. militaris	内源gpd启动子表达密码子优化的Cas9	T7体外转录	分生孢子,ATMT; 原生质体,PMT	NHEJ	ura3,blpR^r	2018^[22]
子囊菌 Ascomycota	竹黄菌 S. bambusicola	内源ef1α启动子表达Cas9	人内源U6启动子	原生质体,PMT	HDR	SbaPKS,hyg^r	2017^[23]