CRISPR/Cas基因编辑及其新兴技术在丝状真菌研究中的系统应用

doi:10.12211/2096-8280.2023-097

合成生物学

• 特约评述 •

CRISPR/Cas基因编辑及其新兴技术在丝状真菌研究中的系统应用

陈盈盈¹, 刘扬¹, 史俊杰¹, 马俊英¹, 鞠建华¹^,²

^1.中国科学院热带海洋生物资源与生态重点实验室（中国科学院南海海洋研究所），广东省海洋药物重点实验室，广东广州 510301
^2.山东大学药学院，天然产物化学生物学教育部重点实验室，山东省基础科学研究中心（药学），山东省高等学校药物化学生物学重点实验室，山东济南 250012

收稿日期:2023-12-01 修回日期:2024-03-08 出版日期:2024-03-12
通讯作者: 马俊英,鞠建华
作者简介:陈盈盈（1986─），女，博士，副研究员。研究方向为真菌次级代谢产物生物合成和代谢调控。 E-mail：yychen@scsio.ac.cn
马俊英（1980─），女，博士，教授，博士生导师。研究方向为结构新颖、活性显著的海洋微生物活性次级代谢产物的生物合成。E-mail：majunying@scsio.ac.cn
鞠建华（1972─），男，博士，教授，博士生导师。研究方向为微生物活性次级代谢产物的发现、生物合成和抗感染、抗肿瘤创新药物研发。E-mail：jju@sdu.edu.cn
基金资助:
国家自然科学基金(22037006);广东省自然科学基金(20023A1515011840);广东省重点领域研发计划(2020B111103005);广东省本土创新创业团队项目(2019BT02Y262)

Progress of the CRISPR/Cas system and the next-generation CRISPR technologies in gene editing for filamentous fungi

Yingying CHEN¹, Yang LIU¹, Junjie SHI¹, Junying MA¹, Jianhua JU¹^,²

^1.CAS Key Laboratory of Tropical Marine Bio-resources and Ecology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，China
^2.School of Pharmaceutical Sciences，Key Laboratory of Chemical Biology （Ministry of Education），Shandong Basic Science Research Center （Pharmacy），Key Laboratory of Medicinal Chemical Biology （Shandong），Shandong University，Jinan 250012，China

Received:2023-12-01 Revised:2024-03-08 Online:2024-03-12
Contact: Junying MA, Jianhua JU

摘要/Abstract

摘要：

丝状真菌（filamentous fungi）具有独特的形态和细胞构造，与人类健康和工农业生产息息相关，对这类生物资源的开发和利用高度依赖高效的基因编辑平台。然而，由于丝状真菌复杂多样的遗传背景，使用传统的基因编辑技术较难实现大范围的基因编辑，极大地妨碍了丝状真菌的遗传学研究。CRISPR/Cas（clustered regularly interspaced short palindromic repeat/CRISPR-associated protein）技术的出现，打破了这一困境，促进了不同种属和不同来源的丝状真菌的基因编辑，为丝状真菌的基础和应用研究带来了革命性的突破。本文简述了CRISPR/Cas系统的作用机理、分类及基于CRISPR的各种新型技术，归纳总结了丝状真菌中现有的CRISPR/Cas9系统功能组分、多种新兴CRISPR/Cas技术在丝状真菌中的应用现状以及海洋真菌中的CRISPR/Cas技术的应用情况。最后，对CRISPR/Cas系统在丝状真菌中应用进展缓慢、编辑效率低和脱靶效应等问题以及针对这些问题的潜在解决方法进行总结和展望，以期为不同类型的丝状真菌基因编辑平台的构建提供参考。

关键词: 丝状真菌, 海洋真菌, CRISPR/Cas, 基因编辑

Abstract:

Filamentous fungi, which harbor distinct morphology and cell structure, play a critical role in human health and industrial/agricultural production. However, the unique characteristics of filamentous fungi make them difficult to be understood, requiring special attention. It's hard to apply common traditional genetic manipulation methods in various filamentous fungi due to the complicated genetic background and special physiological structure, which hampers the genetic manipulation of filamentous fungi. Thus, the development of an efficient gene editing system is essential for exploring biological resources and understanding the biological processes in filamentous fungi. The occurrence of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) system promotes the development of gene editing systems in different species of filamentous fungi derived from various environmental sources, and brings a revolutionary breakthrough in fungal fundamental research and the application of filamentous fungi. In this review, we first briefly introduce the history, working mechanism, and different classifications of the CRISPR/Cas mediated gene editing system. Next, we review the functional components (selective marker, Cas9 and gRNA) of CRISPR/Cas9 and the delivery methods of these components in various filamentous fungi. Then, we systematically summarize the applications of novel CRISPR related technologies, including CRISPR/Cas12, Base-editor, CRISPRa, CRISPRi and CRISPR mediated epigenetic regulation, in filamentous fungi. In particular, the application of CRISPR/Cas mediated gene editing systems in the marine-derived filamentous fungi are highlighted. Finally, we summarize and discuss the slow progress in the application of CRISPR/Cas-based gene editing systems, relative low gene editing efficiency and off- targets effects in filamentous fungi, as well as the potential solutions for the development of novel CRISPR/Cas-based gene editing systems. In general, this review will provide useful guidance for developing an efficient gene editing platform in various filamentous fungi and pave the way for further exploration of secondary metabolites and the establishment of cell factories in filamentous fungi.

Key words: filamentous fungi, marine-derived fungi, CRISPR/Cas, gene editing

中图分类号:

Q812

陈盈盈, 刘扬, 史俊杰, 马俊英, 鞠建华. CRISPR/Cas基因编辑及其新兴技术在丝状真菌研究中的系统应用[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-097.

Yingying CHEN, Yang LIU, Junjie SHI, Junying MA, Jianhua JU. Progress of the CRISPR/Cas system and the next-generation CRISPR technologies in gene editing for filamentous fungi[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-097.

图/表 7

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蛋白名称	蛋白大小（AA）	PAM/TAM 序列	gRNA 大小	剪切位点	参考文献
spCas9	1368	NGG	20 bp	~ 3 bp 5′ of PAM	[12]
FnCas9	1629	NGG	20 bp	~ 3 pb 5′ of PAM	[16]
SaCas9	1053	NNGR RT	21 bp	~ 3 pb 5′ of PAM	[17]
NmCas9	1082	NNNNG ATT	24bp	~ 3 bp 5′ of PAM	[18]
St1Cas9	1121	NNAGA AW	20bp	~ 3 bp 5′ of PAM	[13,19]
St3Cas9	1409	NGGNG	20 bp	~ 3 bp 5′ of PAM	[13,19]
CjCas9	984	NNNNACAC	22 bp	~ 3 bp 5′ of PAM	[20]
CdCas9	1084	NNRHHHY	22 bp	~ 3 bp 5′ of PAM	[21]
GeoCas9	1087	NNNNCRAA	21/22bp	~ 3 bp 5′ of PAM	[22]
AceCas9	1138	NNNCC	20 bp	~ 3 bp 5′ of PAM	[23]
AsCas12a	1307	TTTV	24 bp	~ 19/24 bp 3′ of PAM	[24]
LbCas12a	1228	TTTV	24 bp	~ 19/24 bp 3′ of PAM	[24]
FnCas12a	1307	TTV	24 bp	~ 19/24 bp 3′ of PAM	[25]
Cas12b	1108/1130	TTN	23 bp	~ 17/23 pb 5′ of PAM	[26-27]
Cas12f	~400-600	5′ T/C-rich	20bp/33-39 bp	~ 3/24 bp 3′ of PAM	[28-31]
CasX	978	TTCN	20bp	~ 12 25 bp ′ of 3′ PAM	[32]
TnpB/IscB/IsrB	~400	TTGAT/ATAAA /ATGA/ NNG	15-45bp	~ 3 /12 bp of 5′ TAM ~ 6/21 bp of 3′ TAM	[33-34]
Fz	~500-800	CATA/TTAAN /CCG/TAG	7-21 bp	~ 9/21 bp of 5′ TAM	[35]
Cas14	~400-700	ssDNA	20 bp		[29]
Cas13	~900-1250	RNA targeting	28 bp		[36]

蛋白名称	蛋白大小（AA）	PAM/TAM 序列	gRNA 大小	剪切位点	参考文献
spCas9	1368	NGG	20 bp	~ 3 bp 5′ of PAM	[12]
FnCas9	1629	NGG	20 bp	~ 3 pb 5′ of PAM	[16]
SaCas9	1053	NNGR RT	21 bp	~ 3 pb 5′ of PAM	[17]
NmCas9	1082	NNNNG ATT	24bp	~ 3 bp 5′ of PAM	[18]
St1Cas9	1121	NNAGA AW	20bp	~ 3 bp 5′ of PAM	[13,19]
St3Cas9	1409	NGGNG	20 bp	~ 3 bp 5′ of PAM	[13,19]
CjCas9	984	NNNNACAC	22 bp	~ 3 bp 5′ of PAM	[20]
CdCas9	1084	NNRHHHY	22 bp	~ 3 bp 5′ of PAM	[21]
GeoCas9	1087	NNNNCRAA	21/22bp	~ 3 bp 5′ of PAM	[22]
AceCas9	1138	NNNCC	20 bp	~ 3 bp 5′ of PAM	[23]
AsCas12a	1307	TTTV	24 bp	~ 19/24 bp 3′ of PAM	[24]
LbCas12a	1228	TTTV	24 bp	~ 19/24 bp 3′ of PAM	[24]
FnCas12a	1307	TTV	24 bp	~ 19/24 bp 3′ of PAM	[25]
Cas12b	1108/1130	TTN	23 bp	~ 17/23 pb 5′ of PAM	[26-27]
Cas12f	~400-600	5′ T/C-rich	20bp/33-39 bp	~ 3/24 bp 3′ of PAM	[28-31]
CasX	978	TTCN	20bp	~ 12 25 bp ′ of 3′ PAM	[32]
TnpB/IscB/IsrB	~400	TTGAT/ATAAA /ATGA/ NNG	15-45bp	~ 3 /12 bp of 5′ TAM ~ 6/21 bp of 3′ TAM	[33-34]
Fz	~500-800	CATA/TTAAN /CCG/TAG	7-21 bp	~ 9/21 bp of 5′ TAM	[35]
Cas14	~400-700	ssDNA	20 bp		[29]
Cas13	~900-1250	RNA targeting	28 bp		[36]

分类	筛选标记	表征或筛选条件	应用
抗性基因标记	Hygromycin (hyg)	Hph (编码潮霉素磷酸转移酶), 具有潮霉素抗性	A. niger^[42]， A. fumigatus^[43] et al. 本课题组各种海洋来源菌株
	phosphinothricin (bar)	Bar (编码磷化麦黄酮乙酰转移酶)，具有草胺膦抗性	M. thermophila^[44]， B. bassiana^[45] et al.
	Carboxin (cbx)	点突变的SdhB (编码琥珀酸脱氢酶)可作为米曲霉的筛选标记，以醋酸盐作为碳源，可提高对萎锈灵的敏感性	A. oryzae^[46] et al.
	Neomycin (neo)	Neo (编码氨基糖苷磷酸转移酶)，具有G418 (新霉素衍生物)抗性	P. sojae^[47]， M. thermophila^[48] et al
	Pyrithiamine (ptrA)	点突变的PtrA(编码硫胺素噻唑合成酶基因)，具有吡啶硫胺抗性	A. oryzae^[49]， A. flavus^[50] et al.
	Chlorimuron (sur)	Sur (乙酰乳酸合成酶)具有氯嘧磺隆抗性	K. petricola^[51] et al.
	Fenhexamid (Fen^r)	点突变的 ERG27 (编码酮还原酶)，具有环酰菌胺抗性	B. cinerea^[52] et al.
	Nourseothricin (Nat)	Nat(编码N-乙酰转移酶)，具有诺尔斯菌素抗性	B. cinerea^[52] et al.
营养缺陷型标记	amdS	带有amdS基因的菌株在以乙酰胺为唯一氮源的培养基上能够正常生长。	A. niger^[53], M. thermophila^[48] et al.
	niaD	niaD功能缺陷突变株能够耐受高浓度氯盐	A. oryzae^[49] et al.
	pyrG	pyrG功能缺失的突变株只能在含有尿苷或尿嘧啶的培养基上正常生长	A. niger^[42], A. terreus^[54] et al.
	argB	argB功能缺失的突变株只能在含有精氨酸的培养基上正常生长	A. nidulans^[55]， A. oryzae^[56] et al.
	adeA	adeA功能缺失的突变株只能在含有腺苷酸的培养基上正常生长	A. oryzae^[56] et al.
	ppt1	缺失ppt1功能的突变体只能在添加赖氨酸的培养基上正常生长	F. fujikuroi^[57] et al.
表型报告基因	fcc1	fcc1基因的破坏可导致紫色色素的积累	F. fujikuroi^[57] et al.
	wA	wA突变体形成白色分生孢子	A. oryzae^[49], A. nidulans^[55] et al
	yA	yA突变体形成黄色分生孢子	A. oryzae^[49], A.nidulans^[55] et al
	fwnA	fwnA突变体形成白色分生孢子	A. niger^[58] et al.
	albA	albA突变体形成白色分生孢子	A. niger^[53] et al.
	pkaC	pkaC (编码camp依赖性蛋白激酶的催化亚单位)的破坏可导致平板上的菌落直径减少2~3倍	A. niger^[53] et al.
	creA	creA 敲除菌株产孢能力显著降低	Spiromastix sp. ^[59]et al.
	pksP	pksP敲除菌落具有明显的附白化表型	A. fumigatus^[60] et al.
	CnaA	CnaA功能障碍导致菌丝生长缺陷显著，菌落表型非常小而密集	A. fumigatus^[60] et al.
	ayg1	ayg1 敲除可导致孢子的颜色从黑色变为黄色	A. carbonarius^[61] et al.
	cbh1	破坏cbh1将导致SDS-PAGE凝胶上的主要条带丢失，从而有利于通过成功的基因编辑鉴定菌株	T. reesei^[62] et al.

分类	筛选标记	表征或筛选条件	应用
抗性基因标记	Hygromycin (hyg)	Hph (编码潮霉素磷酸转移酶), 具有潮霉素抗性	A. niger^[42]， A. fumigatus^[43] et al. 本课题组各种海洋来源菌株
	phosphinothricin (bar)	Bar (编码磷化麦黄酮乙酰转移酶)，具有草胺膦抗性	M. thermophila^[44]， B. bassiana^[45] et al.
	Carboxin (cbx)	点突变的SdhB (编码琥珀酸脱氢酶)可作为米曲霉的筛选标记，以醋酸盐作为碳源，可提高对萎锈灵的敏感性	A. oryzae^[46] et al.
	Neomycin (neo)	Neo (编码氨基糖苷磷酸转移酶)，具有G418 (新霉素衍生物)抗性	P. sojae^[47]， M. thermophila^[48] et al
	Pyrithiamine (ptrA)	点突变的PtrA(编码硫胺素噻唑合成酶基因)，具有吡啶硫胺抗性	A. oryzae^[49]， A. flavus^[50] et al.
	Chlorimuron (sur)	Sur (乙酰乳酸合成酶)具有氯嘧磺隆抗性	K. petricola^[51] et al.
	Fenhexamid (Fen^r)	点突变的 ERG27 (编码酮还原酶)，具有环酰菌胺抗性	B. cinerea^[52] et al.
	Nourseothricin (Nat)	Nat(编码N-乙酰转移酶)，具有诺尔斯菌素抗性	B. cinerea^[52] et al.
营养缺陷型标记	amdS	带有amdS基因的菌株在以乙酰胺为唯一氮源的培养基上能够正常生长。	A. niger^[53], M. thermophila^[48] et al.
	niaD	niaD功能缺陷突变株能够耐受高浓度氯盐	A. oryzae^[49] et al.
	pyrG	pyrG功能缺失的突变株只能在含有尿苷或尿嘧啶的培养基上正常生长	A. niger^[42], A. terreus^[54] et al.
	argB	argB功能缺失的突变株只能在含有精氨酸的培养基上正常生长	A. nidulans^[55]， A. oryzae^[56] et al.
	adeA	adeA功能缺失的突变株只能在含有腺苷酸的培养基上正常生长	A. oryzae^[56] et al.
	ppt1	缺失ppt1功能的突变体只能在添加赖氨酸的培养基上正常生长	F. fujikuroi^[57] et al.
表型报告基因	fcc1	fcc1基因的破坏可导致紫色色素的积累	F. fujikuroi^[57] et al.
	wA	wA突变体形成白色分生孢子	A. oryzae^[49], A. nidulans^[55] et al
	yA	yA突变体形成黄色分生孢子	A. oryzae^[49], A.nidulans^[55] et al
	fwnA	fwnA突变体形成白色分生孢子	A. niger^[58] et al.
	albA	albA突变体形成白色分生孢子	A. niger^[53] et al.
	pkaC	pkaC (编码camp依赖性蛋白激酶的催化亚单位)的破坏可导致平板上的菌落直径减少2~3倍	A. niger^[53] et al.
	creA	creA 敲除菌株产孢能力显著降低	Spiromastix sp. ^[59]et al.
	pksP	pksP敲除菌落具有明显的附白化表型	A. fumigatus^[60] et al.
	CnaA	CnaA功能障碍导致菌丝生长缺陷显著，菌落表型非常小而密集	A. fumigatus^[60] et al.
	ayg1	ayg1 敲除可导致孢子的颜色从黑色变为黄色	A. carbonarius^[61] et al.
	cbh1	破坏cbh1将导致SDS-PAGE凝胶上的主要条带丢失，从而有利于通过成功的基因编辑鉴定菌株	T. reesei^[62] et al.

类型	名称	来源	应用
RNA聚合酶II型启动子(组成型启动子)	ptef1	转录延伸因子启动子	A. niger^[83], M. thermophila^[48] et al.
	ptrpC	吲哚甘油磷酸合成酶启动子	N. crassa^[81], S. minima^[84] et al.
	pgpdA	3-磷酸甘油醛脱氢酶的启动子	F. fujikuroi^[57], A. fumigatus^[60] et al.
	ppdc	丙酮酸脱羧酶的启动子	T. reesei^[40], G. lozoyensis^[85] et al.
	pactin	肌动蛋白的启动子	C. globosum^[86]L. maculans^[85] et al.
	phsp70	热休克蛋白的启动子	U. hordei^[87]
	ppkiA	丙酮酸激酶的启动子	A. niger^[88]
	pef1α	人延长因子1α的启动子	S. bambusicola^[89]
	pmbfA	多蛋白桥接因子的启动子	A. niger^[42]
	pcoxA	细胞色素氧化酶的启动子	A. niger^[42]
	p40S	40S 核糖体蛋白S8的启动子	P.rubens^[90]
	pham34	莴苣盘霜霉来源启动子	P.sojae^[47]
	poliC	ATP 合成酶亚基的启动子	B.cinerea^[52]
RNA聚合酶II型启动子(诱导型启动子)	pxylP/xlnA	木聚糖酶的启动子	P. chrysogenum^[73]，A. fumigatus^[91] et al.
	pcbh1	纤维素二糖水解酶I的启动子	T. reesei^[40]
	pamyB	淀粉酶的启动子	A.oryzae^[74]
	ptetON	四环素诱导启动子	A. fumigatus^[92]
	pglaA	α-葡萄糖淀粉酶的启动子	A. niger^[93], T. reesei^[94] et al.
	niiA	硝酸还原酶的启动子	A. fumigatus^[60] et al.
RNA聚合酶III型启动子	u6	人U6微核启动子	A.oryzae^[49], A. richmondensis^[95] et al.
	5S rRNA	5S rRNA基因启动子	A. niger^[53], P. oxalicum^[94] et al.
	tRNA	转录转移核糖核酸启动子	A. niger^[93], A. aculeatus^[96] et al.
	SNR52	核仁小分子RNA 52启动子	N. crassa^[81], A. fumigatus^[43] et al.
体外转录	T7	T7噬菌体衍生启动子	T. reesei^[62], P. chrysogenum^[97] et al.

CRISPR/Cas基因编辑及其新兴技术在丝状真菌研究中的系统应用

Progress of the CRISPR/Cas system and the next-generation CRISPR technologies in gene editing for filamentous fungi

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菌株	CRISPR/Cas12a 存在形式	递送方式	表达策略	编辑效率	参考文献
嗜热毁丝霉 (M.thermophila)	PCR 产物	PEG介导的原生质体转化	Ptef启动子驱动Cas12a的表达；U6启动子驱动gRNA的表达	编辑单基因的效率为90%;编辑多个基因时，单基因发生编辑的效率为13%~41%;	[44]
稻瘟病菌 (M. oryzae )	RNP	PEG介导的原生质体转化	体外表达	50%至100%的编辑效率	[109-110]
棘孢曲霉 (A. aculeatus)	质粒	PEG介导的原生质体转化	Ptef 启动子驱动Cas12a 的表达；U3启动子驱动gRNA的表达	接近100%的编辑效率	[111]
阿舒氏囊霉 (A.gossypii)	质粒	电转	TSA1启动子驱动Cas12a的表达；SNR52启动子驱动gRNA的表达	编辑效率根据靶序列而显著不同（19.2%-77.2%）	[106]
构巢曲霉 (A. nidulans) 黑曲霉 (A. niger)	质粒	PEG介导的原生质体转化	Ptef 启动子驱动Cas12a 的表达；U3启动子驱动gRNA的表达	80%至100%的编辑效率	[112]
嗜热毁丝霉 (T.thermophilus)	RNP/质粒	PEG介导的原生质体转化	Ptef 启动子驱动Cas12a 的表达；U6 启动子驱动gRNA的表达	单基因编辑效率可达100%，双基因编辑效率为40-56%	[113]
米曲霉 (A.oryzae)；酱油曲霉 (A. sojae)	质粒	PEG介导的原生质体转化	Ptef 启动子驱动Cas12a 的表达；gpdA启动子驱动gRNA的表达	在米曲霉中基因编辑效率为60-100%，在酱油霉中基因编辑效率为50-70%	[114-115]

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