CRISPR基因编辑技术在微生物合成生物学领域的研究进展

doi:10.12211/2096-8280.2020-039

摘要/Abstract

摘要：

微生物合成生物学是一门新兴的交叉学科，其主要目的是通过改造或创制微生物细胞，使微生物具有特定的生理功能或生产目标产物，因此需要高效、快速、精准的基因操作工具。CRISPR技术是一种成本低、操作简便、效率高、功能多样的基因编辑技术，近年来被广泛应用于合成生物学、代谢工程和医学研究等领域，极大地促进了这些领域的发展。本文简述了CRISPR基因编辑技术的发展历史及其作用机制，重点介绍了近年来CRISPR/Cas9技术在微生物合成生物学领域研究和应用的进展，列举了CRISPR/Cas9技术在微生物合成生物学中生产目标产品的研究，总结了由CRISPR/Cas9技术衍生出的CRISPR/Cas12a、CRISPR/Cas13等技术在微生物合成生物学领域的研究及应用，提出了CRISPR基因编辑技术现存的PAM依赖性、脱靶效应、安全性和应用广泛性等问题，最后展望了该技术在构建高效微生物细胞工厂生产高附加值化合物的发展前景和创造更多适合生产高附加值产品的底盘生物的研究方向。

关键词: CRISPR, Cas9, 微生物, 合成生物学, 基因编辑

Abstract:

With the increase of global consumption on fossil resources for energy products and chemicals and their consequent impact on environment, construction of microbial cell factories for efficient production of biofuels and bio-based chemicals from renewable sources has gained much attention. Pathway engineering of the hosts, such as over-expression of key genes, disruption of competing pathways and integration of heterologous pathways, plays significant role in fulfilling such a purpose. Successful implementation of these pathway engineering strategies requires efficient and accurate gene editing tools. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a powerful gene editing strategy that was found in prokaryotic organisms such as archaea and bacteria, which provide adaptive immunity against foreign elements. When host cells are infected by viruses, a small sequence of the viral genome is integrated into the CRISPR locus to immunize the host cells, and this small sequence is transcribed into small RNA guide that directs the cleavage of the viral DNA by the Cas nuclease. Inspired by the natural talent, many modified CRISPR systems have been developed to modify genes and genomes, including knock-in, knock-down, large deletions, indels, replacements and chromosomal rearrangements. In this review, we briefly comment on the technical basis and advances in CRISPR-related genome editing tools applied for constructing microbial cell factories, with a focus on the CRISPR-based tools for metabolic engineering of the model organisms E. coli and S. cerevisiae. Furthermore, we highlight major challenges in developing CRISPR tools for multiplex genome editing and sophisticated expression regulation. Finally, we propose future perspectives on the application of CRISPR-based technologies for constructing microbial ecosystems toward high production of desired chemicals. We intend to provide insights and ideas for developing CRISPR-related genome editing tools to better serve the construction of efficient microbial cell factories.

Key words: CRISPR, Cas9, microorganism, synthetic biology, gene editing

中图分类号:

Q812

李洋, 申晓林, 孙新晓, 袁其朋, 闫亚军, 王佳. CRISPR基因编辑技术在微生物合成生物学领域的研究进展[J]. 合成生物学, 2021, 2(1): 106-120.

LI Yang, SHEN Xiaolin, SUN Xinxiao, YUAN Qipeng, YAN Yajun, WANG Jia. Advances of CRISPR gene editing in microbial synthetic biology[J]. Synthetic Biology Journal, 2021, 2(1): 106-120.

图/表 4

图1 CRISPR/Cas9系统切割机制及修复机制图[Cas9蛋白中蓝色代表识别DNA被识别的位点，橙色代表PAM序列，深紫色代表sgRNA上的识别序列；在HDR过程中，浅紫色代表同源序列，绿色代表进行替换的序列，橙色蛋白为RecE或Redα（负责供体DNA单链切割），蓝色蛋白为RecT或Redβ（负责黏性末端的保护以及和基因断裂位点的结合）；在NHEJ过程中，黄色蛋白表示Ku（负责保护断裂位点两侧），橙色蛋白为ligD（负责连接断裂位点）]

Fig. 1 The cleavage and repair mechanism of CRISPR/Cas9 systems（Blue represents the recognized site of DNA, orange represents PAM sequence, and dark purple represents the recognition sequence by sgRNA. During HDR, light purple represents homologous sequences, green represents substituted sequences, orange represents RecE or Red responsible for donor DNA single strand cutting, and blue represents RecT or Red responsible for protecting sticky ends and binding to the breaking sites of genes. During NHEJ, the yellow protein represents Ku responsible for protecting the fracture site, while the orange protein is ligD responsible for connecting to the fracture site)

图2 Cas12a和Cas13a系统作用机制[Cas12a中橙色序列为TTTN的PAM序列，Cas13a中蓝色序列为PFS（Protospacer flanking site）序列]

Fig. 2 Working mechanism of Cas12a and Cas13a systems[Orange in Cas12a represents the PAM sequence of TTTN, and blue in Cas13a represents PFS (protospacer flanking site) sequence]

表1 CRISPR技术在微生物合成生物学中生产目标产品的研究

Tab. 1 Applications of CRISPR-based technologies for the construction of microbial cell factories

物种	系统	产品	编辑后取得效果	参考文献
E. coli	Cas9	β-胡萝卜素	2.0 g/L	[39]
		脂肪酸酯	32 g/L	[50]
		尿苷	5.6 g/L	[43]
		正丁醇	4.32 g/L	[49]
		己二酸	68 g/L	[51]
		庚酸	增强庚酸耐受性	[52]
S. cerevisiae	Cas9	纤维二糖	速率提高10倍	[61]
		甲羟戊酸	比野生型高41倍	[65]
		(R,R)-(-)-2,3-丁二醇	12.51 g/L	[62]
		β-胡萝卜素	提高了3倍	[66]
		紫杉二烯	提高了25倍	[67]
		乙醇	提高了74.7%	[68-70]
		脂肪酸	提高了30倍	[64]
		3-羟基丙酸	11.4 g/L	[71]
		萜类	未具体说明	[72]
		青蒿素类	740 mg/L	[73]
Actinoplanes sp.	Cas9	阿卡波糖	提高了纯度	[79]
Streptomyces rimosus	Cas9	土霉素	增加了36.8%	[80]
Saccharopolyspora erythraea	Cas9	红霉素	比野生型高80.3%	[81]
Corynebacterium glutamicum	Cas12a	半胱氨酸；丝氨酸	半胱氨酸提高3.7倍，丝氨酸提高2.5倍	[101]

表2 CRISPR/Cas9、Cas12a、Cas13a对比总结

Tab. 2 The comparison and summary of CRISPR/Cas9， Cas12a and Cas13a

技术	特点						优势	缺陷
技术	类型	标靶	是否需要tracrRNA	PAM	间隔区间长度	切割结构域	优势	缺陷
CRISPR/Cas9	II	dsDNA	是	NGG-3'	20	HNH和RuvC	成本低；打靶效率高；操作容易；应用广泛	PAM（NGG）依赖性；依然存在脱靶效应；可控性差；某些物种编辑效率低；内源性系统的干扰；对细胞致死性
CRISPR/Cas12a（Cpf1）	V	dsDNA	否	5'-TTTN	23	RuvC	切割产生黏性末端；低毒性；蛋白分子较小；在某些物种中编辑效率高于Cas9系统；不需要tracrRNA	研究不成熟
CRISPR/Cas13a（C2c2）	VI	ssRNA	否	PFS	—	HEPN	对RNA干扰和编辑；脱靶率低于Cas9系统；蛋白分子小	研究较少；应用领域目前较窄

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