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.

Yang LI, Xiaolin SHEN, Xinxiao SUN, Qipeng YUAN, Yajun YAN, Jia WANG. 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

参考文献 119

1	THOMASON L C, COSTANTINO N, COURT D L. E. coli genome manipulation by P1 transduction[J]. Current Protocols in Molecular Biology, 2007, 79(1): 1.17.1-1.17.8.
2	PORTEUS M H, CARROLL D. Gene targeting using zinc finger nucleases[J]. Nature Biotechnology, 2005, 23(8): 967-973.
3	LEE J J, CROOK N, SUN J, et al. Improvement of lactic acid production in Saccharomyces cerevisiae by a deletion of ssb1 [J]. Journal of Industrial Microbiology and Biotechnology, 2016, 43(1): 87-96.
4	JOUNG J K, SANDER J D. TALENs: a widely applicable technology for targeted genome editing [J]. Nature Reviews Molecular Cell Biology, 2013, 14(1): 49-55.
5	TIAN P F, WANG J, SHEN X L, et al. Fundamental CRISPR-Cas9 tools and current applications in microbial systems [J]. Synthetic and Systems Biotechnology, 2017, 2(3): 219-225.
6	汪莲, 王浩君, 罗云孜. CRISPR技术在微生物合成生物学中的应用[J]. 生命科学, 2019, 31(5):493-507.
	WANG L, WANG H J, LUO Y Z. Applications of CRISPR technology in microbial synthetic biology[J]. Chinese Bulletin of Life Sciences, 2019, 31(5): 493-507.
7	KOONIN E V, MAKAROVA K S, ZHANG F. Diversity, classification and evolution of CRISPR-Cas systems [J]. Current Opinion in Microbiology, 2017, 37: 67-78.
8	HORII T. Genome engineering using the CRISPR/Cas system [J]. World Journal of Medical Genetics, 2014, 4(3): 69-76.
9	张昆, 陈景超, 李祎, 等. CRISPR/Cas9技术在微生物研究中的应用进展[J]. 微生物学通报, 2018, 45(2): 451-464.
	ZHANG K, CHEN J C, LI Y, et al. Application progress of CRISPR/Cas9 technology in microbiological research[J]. Microbiology China, 2018, 45(2): 451-464.
10	PAN M, BARRANGOU R. Combining omics technologies with CRISPR-based genome editing to study food microbes [J]. Current Opinion in Biotechnology, 2020, 61: 198-208.
11	ISHINO Y, SHINAGAWA H, MAKINO K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product [J]. Journal of Bacteriology, 1987, 169(12): 5429-5433.
12	MOJICA F J, DIEZ-VILLASENOR C, SORIA E, et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria [J]. Molecular Microbiology, 2000, 36(1): 244-246.
13	JANSEN R, EMBDEN J D, GAASTRA W, et al. Identification of genes that are associated with DNA repeats in prokaryotes [J]. Molecular Microbiology, 2002, 43(6): 1565-1575.
14	BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes [J]. Science, 2007, 315(5819): 1709-1712.
15	BROUNS S J, JORE M M, LUNDGREN M, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes [J]. Science, 2008, 321(5891): 960-964.
16	MARRAFFINI L A, SONTHEIMER E J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA [J]. Science, 2008, 322(5909): 1843-1845.
17	DELTCHEVA E, CHYLINSKI K, SHARMA C M, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Ⅲ [J]. Nature, 2011, 471(7340): 602-607.
18	JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J]. Science, 2012, 337(6096): 816-821.
19	CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems [J]. Science, 2013, 339(6121): 819-823.
20	GARST A D, BASSALO M C, PINES G, et al. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering [J]. Nature Biotechnology, 2017, 35(1): 48-55.
21	WANG T M, GUAN C G, GUO J H, et al. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance [J]. Nature Communications, 2018, 9(1): 2475.
22	LIAN J Z, SCHULTZ C, CAO M F, et al. Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping [J]. Nature Communications, 2019, 10(1): 5794.
23	LEE H H, OSTROV N, WONG B G, et al. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi [J]. Nature Microbiology, 2019, 4(7): 1105-1113.
24	SHEN B, ZHANG J, WU H Y, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting [J]. Cell Research, 2013, 23(5): 720-723.
25	HWANG W Y, FU Y F, REYON D, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system [J]. Nature Biotechnology, 2013, 31(3): 227-229.
26	FRIEDLAND A E, TZUR Y B, ESVELT K M, et al. Heritable genome editing in C. elegansvia a CRISPR-Cas9 system [J]. Nature Methods, 2013, 10(8): 741-743.
27	JIANG W Z, ZHOU H B, BI H H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice[J]. Nucleic Acids Research, 2013, 41(20): e188.
28	NODVIG C S, NIELSEN J B, KOGLE M E, et al. A CRISPR-Cas9 system for genetic engineering of filamentous fungi [J]. PLoS One, 2015, 10(7): e0133085.
29	CHOI K R, LEE S Y. CRISPR technologies for bacterial systems: current achievements and future directions [J]. Biotechnology Advances, 2016, 34(7): 1180-1209.
30	LIANG F, HAN M G, ROMANIENKO P J, et al. Homology-directed repair is a major double-strand break repair pathway in mammalian cells [J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(9): 5172-5177.
31	LIEBER M R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway [J]. Annual Review of Biochemistry, 2010, 79: 181-211.
32	BARRANGOU R. CRISPR-Cas systems and RNA-guided interference [J]. Wiley Interdisciplinary Reviews-RNA, 2013, 4(3): 267-278.
33	SHMAKOV S, ABUDAYYEH O O, MAKAROVA K S, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems [J]. Molecular Cell, 2015, 60(3): 385-397.
34	MAKAROVA K S, HAFT D H, BARRANGOU R, et al. Evolution and classification of the CRISPR-Cas systems [J]. Nature Reviews Microbiology, 2011, 9(6): 467-477.
35	ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system [J]. Cell, 2015, 163(3): 759-771.
36	MARRAFFINI L A, SONTHEIMER E J. Self versus non-self discrimination during CRISPR RNA-directed immunity [J]. Nature, 2010, 463(7280): 568-571.
37	PONTRELLI S, CHIU T Y, LAN E I, et al. Escherichia coli as a host for metabolic engineering [J]. Metabolic Engineering, 2018, 50: 16-46.
38	JIANG W Y, BIKARD D, COX D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems [J]. Nature Biotechnology, 2013, 31(3): 233-239.
39	LI Y F, LIN Z Q, HUANG C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing [J]. Metabolic Engineering, 2015, 31: 13-21.
40	JIANG Y, CHEN B, DUAN C L, et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system [J]. Applied and Environmental Microbiology, 2015, 81(7): 2506-2514.
41	ZHAO D D, YUAN S L, XIONG B, et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9 [J]. Microbial Cell Factories, 2016, 15(1): 205.
42	CHUNG M E, YEH I H, SUNG L Y, et al. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9 [J]. Biotechnology and Bioengineering, 2017, 114(1): 172-183.
43	LI Y F, YAN F Q, WU H Y, et al. Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli [J]. Journal of Industrial Microbiology and Biotechnology, 2019, 46(1): 81-90.
44	WILSON T E, TOPPER L M, PALMBOS P L. Non-homologous end-joining: bacteria join the chromosome breakdance [J]. Trends in Biochemical Sciences, 2003, 28(2): 62-66.
45	ZHENG X, LI S Y, ZHAO G P, et al. An efficient system for deletion of large DNA fragments in Escherichia colivia introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis [J]. Biochemical and Biophysical Research Communications, 2017, 485(4): 768-774.
46	HUANG C Y, DING T T, WANG J G, et al. CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli [J]. Applied Microbiology and Biotechnology, 2019, 103(20): 8497-8509.
47	KITNEY R I. Synthetic biology-engineering biologically-based devices and systems [M]. Berlin: Springer, 2007.
48	夏军, 郑明刚, 王玲, 等. 运用CRISPR/Cas系统敲除大肠杆菌磷酸烯醇式丙酮酸羧化酶基因及其对脂肪酸代谢的影响[J]. 微生物学通报, 2016, 43(8): 1864-1871.
	XIA J, ZHENG M G, WANG L, et al. Knocking out phosphoenolpyruvate carboxylase gene by CRISPR/Cas and its influence on fatty acid metabolism in Escherichia coli[J]. Microbiology China, 2016, 43(8): 1864-1871.
49	ABDELAAL A S, JAWED K, YAZDANI S S. CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium [J]. Journal of Industrial Microbiology and Biotechnology, 2019, 46(7): 965-975.
50	JUNG H R, YANG S Y, MOON Y M, et al. Construction of efficient platform Escherichia coli strains for polyhydroxyalkanoate production by engineering branched pathway [J]. Polymers (Basel), 2019, 11(3).
51	ZHAO M, HUANG D X, ZHANG X J, et al. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway [J]. Metabolic Engineering, 2018, 47: 254-262.
52	SEO J H, BAEK S W, LEE J, et al. Engineering Escherichia coli BL21 genome to improve the heptanoic acid tolerance by using CRISPR-Cas9 system [J]. Biotechnology and Bioprocess Engineering, 2017, 22(3): 231-238.
53	OU X Y, WU X L, PENG F, et al. Metabolic engineering of a robust Escherichia coli strain with a dual protection system [J]. Biotechnology and Bioengineering, 2019, 116(12): 3333-3348.
54	LEE S M, JELLISON T, ALPER H S. Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields [J]. Biotechnology for Biofuels, 2014, 7(1): 122.
55	LIAN J Z, MISHRA S, ZHAO H M. Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications [J]. Metabolic Engineering, 2018, 50: 85-108.
56	PADDON C J, WESTFALL P J, PITERA D J, et al. High-level semi-synthetic production of the potent antimalarial artemisinin [J]. Nature, 2013, 496(7446): 528-532.
57	MANS R, ROSSUM H M VAN, WIJSMAN M, et al. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae [J]. FEMS Yeast Research, 2015, 15(2): fov004.
58	DICARLO J E, NORVILLE J E, MALI P, et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems [J]. Nucleic Acids Research, 2013, 41(7): 4336-4343.
59	HORWITZ A A, WALTER J M, SCHUBERT M G, et al. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas [J]. Cell Systems, 2015, 1(1): 88-96.
60	BAO Z H, XIAO H, LIANG J, et al. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae [J]. ACS Synthetic Biology, 2015, 4(5): 585-594.
61	RYAN O W, SKERKER J M, MAURER M J, et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system [J]. eLife, 2014, 3: e03703.
62	SHI S B, LIANG Y Y, ZHANG M M, et al. A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae [J]. Metabolic Engineering, 2016, 33: 19-27.
63	BAO Z H, HAMEDIRAD M, XUE P, et al. Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision [J]. Nature Biotechnology, 2018, 36(6): 505-508.
64	ZHANG Y P, WANG J, WANG Z B, et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae [J]. Nature Communications, 2019, 10(1): 1053.
65	JAKOČIŪNAS T, BONDE I, HERRGARD M, et al. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae [J]. Metabolic Engineering, 2015, 28: 213-222.
66	LIAN J Z, HAMEDIRAD M, HU S M, et al. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system [J]. Nature Communications, 2017, 8(1): 1688.
67	REIDER APEL A, D'ESPAUX L, WEHRS M, et al. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae [J]. Nucleic Acids Research, 2017, 45(1): 496-508.
68	XUE T, LIU K, CHEN D, et al. Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae [J]. World Journal of Microbiology and Biotechnology, 2018, 34(10): 154.
69	LIU K, YUAN X, LIANG L M, et al. Using CRISPR/Cas9 for multiplex genome engineering to optimize the ethanol metabolic pathway in Saccharomyces cerevisiae [J]. Biochemical Engineering Journal, 2019, 145: 120-126.
70	YANG P Z, WU Y, ZHENG Z, et al. CRISPR-Cas9 approach constructing cellulase sestc-engineered Saccharomyces cerevisiae for the production of orange peel ethanol [J]. Frontiers in Microbiology, 2018, 9: 2436.
71	TAKAYAMA S, OZAKI A, KONISHI R, et al. Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe [J]. Microbial Cell Factories, 2018, 17(1): 176.
72	WANG C L, LIWEI M, PARK J B, et al. Microbial Platform for Terpenoid Production: Escherichia coli and Yeast [J]. Frontiers in Microbiology, 2018, 9: 2460.
73	AI L M, GUO W W, CHEN W, et al. The gal80 deletion by CRISPR-Cas9 in engineered Saccharomyces cerevisiae produces artemisinic acid without galactose induction [J]. Current Microbiology, 2019, 76(11): 1313-1319.
74	XIE Z X, LI B Z, MITCHELL L A, et al. "Perfect" designer chromosome V and behavior of a ring derivative [J]. Science, 2017, 355(6329): eaaf4704.
75	SHAO Y Y, LU N, CAI C, et al. A single circular chromosome yeast [J]. Cell Research, 2019, 29(1): 87-89.
76	SHAO Y Y, LU N, WU Z F, et al. Creating a functional single-chromosome yeast [J]. Nature, 2018, 560(7718): 331-335.
77	GENILLOUD O. Actinomycetes: still a source of novel antibiotics [J]. Natural Product Reports, 2017, 34(10): 1203-1232.
78	COBB R E, SHAO Y Y, LU N, WU Z F. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system [J]. ACS Synthetic Biology, 2015, 4(6): 723-728.
79	WOLF T, GREN T, THIEME E, et al. Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System [J]. Journal of Biotechnology, 2016, 231: 122-128.
80	JIA H Y, ZHANG L M, WANG T T, et al. Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus [J]. Microbiology, 2017, 163(8): 1148-1155.
81	LIU Y, WEI W P, YE B C. High GC content Cas9-mediated genome-editing and biosynthetic gene cluster activation in Saccharopolyspora erythraea [J]. ACS Synthetic Biology, 2018, 7(5): 1338-1348.
82	LOW Z J, PANG L M, DING Y, et al. Identification of a biosynthetic gene cluster for the polyene macrolactam sceliphrolactam in a Streptomyces strain isolated from mangrove sediment [J]. Scientific Reports, 2018, 8(1): 1594.
83	GUTIERREZ-CORREA M, LUDENA Y, RAMAGE G, et al. Recent advances on filamentous fungal biofilms for industrial uses [J]. Applied Biochemistry and Biotechnology, 2012, 167(5): 1235-1253.
84	MEYER V. Genetic engineering of filamentous fungi - progress, obstacles and future trends [J]. Biotechnology Advances, 2008, 26(2): 177-185.
85	LIU R, CHEN L, JIANG Y P, et al. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system [J]. Cell Discovery, 2015, 1: 15007.
86	POHL C, KIEL J A, DRIESSEN A J, et al. CRISPR/Cas9 based genome editing of Penicillium chrysogenum [J]. ACS Synthetic Biology, 2016, 5(7): 754-764.
87	QIN H, XIAO H, ZOU G, et al. CRISPR-Cas9 assisted gene disruption in the higher fungus Ganoderma species [J]. Process Biochemistry, 2017, 56: 57-61.
88	JIANG D W, ZHU W, WANG Y C, et al. Molecular tools for functional genomics in filamentous fungi: recent advances and new strategies [J]. Biotechnology Advances, 2013, 31(8): 1562-1574.
89	KATAYAMA T, TANAKA Y, OKABE T, et al. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae [J]. Biotechnology Letters, 2016, 38(4): 637-642.
90	MATSU-URA T, BAEK M, KWON J, et al. Efficient gene editing in Neurospora crassa with CRISPR technology [J]. Fungal Biology and Biotechnology, 2015, 2: 4.
91	ARAZOE T, MIYOSHI K, YAMATO T, et al. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus [J]. Biotechnology and Bioengineering, 2015, 112(12): 2543-2549.
92	VYAS V K, BARRASA M I, FINK G R. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families [J]. Science Advances, 2015, 1(3): e1500248.
93	TAO P, WU X R, TANG W C, et al. Engineering of bacteriophage T4 genome using CRISPR-Cas9 [J]. ACS Synthetic Biology, 2017, 6(10): 1952-1961.
94	RAO V B, BLACK L W. Structure and assembly of bacteriophage T4 head [J]. Virology Journal, 2010, 7: 356.
95	HOSHIGA F, YOSHIZAKI K, TAKAO N, et al. Modification of T2 phage infectivity toward Escherichia coli O157:H7 via using CRISPR/Cas9 [J]. FEMS Microbiology Letters, 2019, 366(4).
96	MAKAROVA K S, WOLF Y I, ALKHNBASHI O S, et al. An updated evolutionary classification of CRISPR-Cas systems [J]. Nature Reviews Microbiology, 2015, 13(11): 722-736.
97	LI Z H, LIU M, LYU X M, et al. CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae [J]. Journal of Basic Microbiology, 2018, 58(12): 1100-1104.
98	LI Z H, LIU M, WANG F Q, et al. Cpf1-assisted efficient genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae [J]. Biotechnology Letters, 2018, 40(8): 1253-1261.
99	SWIAT M A, DASHKO S, RIDDER M DEN, et al. FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae [J]. Nucleic Acids Research, 2017, 45(21): 12585-12598.
100	LI L, WEI K K, ZHENG G S, et al. CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces [J]. Applied and Environmental Microbiology, 2018, 84(18).
101	LIU W, TANG D D, WANG H J, et al. Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a [J]. Applied Microbiology and Biotechnology, 2019, 103(21/22): 8911-8922.
102	ZHOU J, ZHU T C, CAI Z, et al. From cyanochemicals to cyanofactories: a review and perspective [J]. Microbial Cell Factories, 2016, 15: 2.
103	LI H, SHEN C R, HUANG C H, et al. CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production [J]. Metabolic Engineering, 2016, 38: 293-302.
104	UNGERER J, PAKRASI H B. Cpf1 is a versatile tool for CRISPR genome editing across diverse species of Cyanobacteria [J]. Scientific Reports, 2016, 6: 39681.
105	NIU T C, LIN G M, XIE L R, et al. Expanding the potential of CRISPR-Cpf1-based genome editing technology in the Cyanobacterium Anabaena PCC 7120 [J]. ACS Synthetic Biology, 2019, 8(1): 170-180.
106	COX D B T, GOOTENBERG J S, ABUDAYYEH O O, et al. RNA editing with CRISPR-Cas13 [J]. Science, 2017, 358(6366): 1019-1027.
107	KONERMANN S, LOTFY P, BRIDEAU N J, et al. Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors [J]. Cell, 2018, 173(3): 665-676.
108	SMARGON A A, COX D B T, PYZOCHA N K, et al. Cas13b is a type Ⅵ-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28 [J]. Molecular Cell, 2017, 65(4): 618-630.
109	HARRINGTON L B, BURSTEIN D, CHEN J S, et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes [J]. Science, 2018, 362(6416): 839-842.
110	HIDALGO-CANTABRANA C, BARRANGOU R. Characterization and applications of type Ⅰ CRISPR-Cas systems[J]. Biochemical Society Transactions, 2020, 48(1): 15-23.
111	DOLAN A E, HOU Z G, XIAO Y B, et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type Ⅰ CRISPR-Cas[J]. Molecular Cell, 2019, 74(5): 936-950.
112	HIDALGO-CANTABRANA C, GOH Y J, PAN M, et al. Genome editing using the endogenous type Ⅰ CRISPR-Cas system in Lactobacillus crispatus [J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(32): 15774-15783.
113	WANG F, WANG L R, ZOU X, et al. Advances in CRISPR-Cas systems for RNA targeting, tracking and editing [J]. Biotechnology Advances, 2019, 37(5): 708-729.
114	OZCAN A, PAUSCH P, LINDEN A, et al. Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum [J]. Nature Microbiology, 2019, 4(1): 89-96.
115	RAN F A, HSU P D, LIN C Y, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity [J]. Cell, 2013, 154(6): 1380-1389.
116	MORENO-MATEOS M A, VEJNAR C E, BEAUDOIN J D, et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo [J]. Nature Methods, 2015, 12(10): 982-988.
117	GUO J H, WANG T M, GUAN C G, et al. Improved sgRNA design in bacteria via genome-wide activity profiling [J]. Nucleic Acids Research, 2018, 46(14): 7052-7069.
118	LIN L, HE X B, ZHAO T Y, et al. Engineering the direct repeat sequence of crRNA for optimization of FnCpf1-mediated genome editing in human cells [J]. Molecular Therapy, 2018, 26(11): 2650-2657.
119	SHIN J Y, JIANG F G, LIU J J, et al. Disabling Cas9 by an anti-CRISPR DNA mimic [J]. Science Advances, 2017, 3(7): e1701620.

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