Research progress of genome editing technologies for industrial filamentous fungi

doi:10.12211/2096-8280.2020-073

Abstract

Abstract:

Filamentous fungi, which are a large and diverse group of multicellular eukaryotic microorganisms existing in nature universally, have played pivotal roles in biotechnology as the prominent producers of enzymes, organic acids and antibiotics. Filamentous fungi are also important decomposers that contribute to the biological carbon cycle of plant biomass. Genetic engineering is a powerful approach for researchers not only to elucidate the gene function in filamentous fungi, but also to improve their production levels and minimize unwanted by-product formation. However, the efficiency of homologous integration in filamentous fungi is very low using classical genetic approaches. Due to the complicated growth and lifestyle of filamentous fungi, the development of genetic tools and gene editing is relatively slow, which hinders the basic research and biotechnological development of filamentous fungi. In recent years, genome editing technologies based on programmable nucleases have been developed as the powerful gene engineering tools in a wide variety of organisms. The most rapidly developed and wildly used technology is the class of RNA-guided Cas9 nuclease known as the adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). The Cas9-sgRNA complex binds to the corresponding target site of the protospacer in a genome, and the specifically induces double-strand breaks. These breaks can be used as a basis for site-specific mutagenesis mediated by non-homologous end-joining or for the introduction of precise mutation or integration via homology-directed repair. CRISPR-Cas systems have recently enabled a wide range of applications for genome editing in many organisms. Remarkably, and in just the past few years, the CRISPR-Cas9 system has emerged as a more efficient strategy for gene editing in filamentous fungi. In this review, we describe the research progress of three most widely used genome editing systems, including the development of CRISPR-Cas technology and its applications in filamentous fungi, especially recent advances in industrial filamentous fungi. Finally, we give the perspectives for the CRISPR-Cas technology and its derivative systems for genomic editing in filamentous fungi.

Key words: genome editing, nucleases, CRISPR-Cas system, filamentous fungi, industrial filamentous fungi

摘要：

丝状真菌（filamentous fungi）是广泛存在于自然界中的一类多细胞真核微生物，是酶制剂、有机酸、抗生素的核心生产体系，在生物技术中发挥着非常重要的作用。由于丝状真菌的生长发育较为复杂，其遗传体系和基因组编辑技术发展相对较慢，妨碍了丝状真菌基础研究和开发利用的快速发展。近年来，核酸酶介导的基因组编辑技术已经发展成为一种功能强大的基因组编辑工具，在生物技术领域的应用得到广泛关注，其中RNA介导的CRISPR系统（clustered regularly interspaced short palindromic repeats）已经成为新一代基因组编辑技术体系。文章将对目前使用最为广泛的3种基因编辑系统，包括CRISPR-Cas技术的发展历程进行概述，对CRISPR-Cas技术在丝状真菌基因组编辑中的研发进行系统介绍，包括在工业丝状真菌中的研发应用，例如鉴定次级代谢产物的关键基因并提高次级代谢物的生产能力、将外源基因定点整合到基因组上从而提高异源蛋白的表达、遗传重构蛋白分泌途径提高工业酶的产量等，最后对CRISPR-Cas基因组编辑技术及其衍生系统在丝状真菌的真菌基因功能研究、代谢途径重构、精确表达调控、蛋白定向进化以及高性能底盘构建等方面进行了展望。

关键词: 基因组编辑技术, 核酸酶, CRISPR-Cas系统, 丝状真菌, 工业丝状真菌

CLC Number:

Q812

Qian LIU, Jingen LI, Chenyang ZHANG, Fangya LI, Chaoguang TIAN. Research progress of genome editing technologies for industrial filamentous fungi[J]. Synthetic Biology Journal, 2021, 2(2): 256-273.

刘倩, 李金根, 张晨阳, 李芳雅, 田朝光. 工业丝状真菌基因组编辑技术研究进展[J]. 合成生物学, 2021, 2(2): 256-273.

Figures/Tables 3

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丝状真菌	Cas 类型	Cas启动子	sgRNA启动子	靶标基因	编辑效率	参考文献
黑曲霉	Cas9	P_tef1	P_gpdA	albA	高效	[52]
		P_tef1	Af_U6-1p	albA	高效	[53]
		P_tef1	PanU6	albA	79%	[54]
		P_gpdA	5S rRNA	alba, fum5, fum1	高达100%	[55]
		P_tef1	AoU6, AfU6	fwnA, amyA, glaA	单基因高达80%	[56-58]
		P_tef1, P_gpdA, P_glaA	Anp	pyrG, moc, laeA	高达97.2%	[59]
	LbCpf1	P_tef1	Af_U3p	albA	80%	[60]
米曲霉	Cas9	P_amyB	米曲霉U6p	wA, pyrG, yA	10%～100%	[61]
米曲霉	Cas9	P_Aotef1, AMA1-自主复制质粒	PU6p，AMA1-自主复制质粒	wA, pyrG, yA	55.6%～100%	[62]
里氏木霉	Cas9	P_pdc, P_cbh1	体外转录合成	ura5, lae1, vib1, clr2	单基因高达93%～100%；多基因同时编辑效率4.2%～45%	[63]
		体外转录合成	体外转录合成	cbh-1	40%	[64]
		P_pdc	里氏木霉PU61, PU62	ura5	高效	[65]
		体外转录合成	体外转录合成	Trura5,Trlae1,Trcbh1, Trcbh2, Treg1	56.52%～100%	[66]
产黄青霉	Cas9	P_xyl或体外转录合成	U6, 145 tRNA, Utp25或体外转录合成	pks17, roqA, lovF,pcbAB, penDE, hcpA	高达60%～100%	[67, 68]
嗜热毁丝霉	Cas9	P_tef1	嗜热毁丝霉MtU6p	amd S, cre1, res1, alp1, gh1-1, rca1, hcr1, ap3, prk6	单基因高达95%；双、三、四基因同时编辑效率22%～70%	[75]
	SpCas9, FnCpf1, AsCpf1	P_tef1或体外转录合成	U6p或体外转录合成	pks4.2, alp1, snc1, ptf1	单基因高达100%；多基因同时编辑时单基因效率5%～100%	[76]
	AsCas12a	P_tef1	MtU6p	cre1, res1, alp1, gh1-1, rca1,neo, bar, hcr1, ap3, prk6	单基因高达90%；三、四基因同时编辑效率22%～41%	[77]
棉阿舒囊霉	SpCas9	酿酒酵母P_tef1	P_snr52	ade2, A754, fmp27	36%～80%	[78]
棉阿舒囊霉	LbCpf1	P_tsa1	P_snr52	his3, ade2, trp1, leu2, ura3	多基因编辑效率10.4%～77.2%	[79]

丝状真菌	Cas 类型	Cas启动子	sgRNA启动子	靶标基因	编辑效率	参考文献
黑曲霉	Cas9	P_tef1	P_gpdA	albA	高效	[52]
		P_tef1	Af_U6-1p	albA	高效	[53]
		P_tef1	PanU6	albA	79%	[54]
		P_gpdA	5S rRNA	alba, fum5, fum1	高达100%	[55]
		P_tef1	AoU6, AfU6	fwnA, amyA, glaA	单基因高达80%	[56-58]
		P_tef1, P_gpdA, P_glaA	Anp	pyrG, moc, laeA	高达97.2%	[59]
	LbCpf1	P_tef1	Af_U3p	albA	80%	[60]
米曲霉	Cas9	P_amyB	米曲霉U6p	wA, pyrG, yA	10%～100%	[61]
米曲霉	Cas9	P_Aotef1, AMA1-自主复制质粒	PU6p，AMA1-自主复制质粒	wA, pyrG, yA	55.6%～100%	[62]
里氏木霉	Cas9	P_pdc, P_cbh1	体外转录合成	ura5, lae1, vib1, clr2	单基因高达93%～100%；多基因同时编辑效率4.2%～45%	[63]
		体外转录合成	体外转录合成	cbh-1	40%	[64]
		P_pdc	里氏木霉PU61, PU62	ura5	高效	[65]
		体外转录合成	体外转录合成	Trura5,Trlae1,Trcbh1, Trcbh2, Treg1	56.52%～100%	[66]
产黄青霉	Cas9	P_xyl或体外转录合成	U6, 145 tRNA, Utp25或体外转录合成	pks17, roqA, lovF,pcbAB, penDE, hcpA	高达60%～100%	[67, 68]
嗜热毁丝霉	Cas9	P_tef1	嗜热毁丝霉MtU6p	amd S, cre1, res1, alp1, gh1-1, rca1, hcr1, ap3, prk6	单基因高达95%；双、三、四基因同时编辑效率22%～70%	[75]
	SpCas9, FnCpf1, AsCpf1	P_tef1或体外转录合成	U6p或体外转录合成	pks4.2, alp1, snc1, ptf1	单基因高达100%；多基因同时编辑时单基因效率5%～100%	[76]
	AsCas12a	P_tef1	MtU6p	cre1, res1, alp1, gh1-1, rca1,neo, bar, hcr1, ap3, prk6	单基因高达90%；三、四基因同时编辑效率22%～41%	[77]
棉阿舒囊霉	SpCas9	酿酒酵母P_tef1	P_snr52	ade2, A754, fmp27	36%～80%	[78]
棉阿舒囊霉	LbCpf1	P_tsa1	P_snr52	his3, ade2, trp1, leu2, ura3	多基因编辑效率10.4%～77.2%	[79]

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