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 ›› 2024, Vol. 5 ›› Issue (3): 672-693.DOI: 10.12211/2096-8280.2023-097

• 特约评述 • 上一篇

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.中国科学院南海海洋研究所，中国科学院热带海洋生物资源与生态重点实验室，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:2024-06-30 发布日期:2024-07-12
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)

CRISPR/Cas systems and their applications in gene editing with 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，Guangdong，China
^2.Key Laboratory of Chemical Biology （Ministry of Education），Shandong Basic Science Research Center （Pharmacy），Key Laboratory of Medicinal Chemical Biology （Shandong），School of Pharmaceutical Sciences，Shandong University，Ji’nan 250012，Shandong，China

Received:2023-12-01 Revised:2024-03-08 Online:2024-06-30 Published:2024-07-12
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 present distinct morphology and cell structure, play a critical role in human health as well as industrial and agricultural production. However, the unique characteristics of filamentous fungi make them difficult to be manipulated with traditional genetic engineering methods. Thus, the development of an efficient gene editing system is essential for exploring biological resources and understanding metabolic processes in filamentous fungi. The development of the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) system promotes more efficient and effective gene editing in different species, and brings a revolutionary breakthrough in fungal fundamental research and applications. In this review, we first briefly introduce the history, working mechanism, and classifications of the CRISPR/Cas mediated gene editing system. Next, we comment the functional components of CRISPR/Cas9 such as selective marker, Cas9 and gRNA and the delivery methods of these components in various filamentous fungi. Furthermore, we systematically discuss the applications of CRISPR related technologies, including CRISPR/Cas12, base-editor, CRISPRa, CRISPRi and CRISPR mediated epigenetic regulation, in the genetic engineering of filamentous fungi, particularly in marine-derived filamentous fungi. Finally, we address challenges with relative low gene editing efficiency and off-targets effects in engineering filamentous fungi, and highlight the potential solutions for developing novel CRISPR/Cas-based gene editing systems. This review can provide guidance for developing an efficient gene editing platform in filamentous fungi and pave the way for further exploration of the secondary metabolites and establishment of robust fungal cell factories.

Key words: filamentous fungi, marine-derived fungi, CRISPR/Cas systems, 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, 2024, 5(3): 672-693.

Yingying CHEN, Yang LIU, Junjie SHI, Junying MA, Jianhua JU. CRISPR/Cas systems and their applications in gene editing with filamentous fungi[J]. Synthetic Biology Journal, 2024, 5(3): 672-693.

图/表 7

参考文献 147

1	MCCLUSKEY K, BAKER S E. Diverse data supports the transition of filamentous fungal model organisms into the post-genomics era[J]. Mycology, 2017, 8(2): 67-83.
2	ETXEBESTE O, ESPESO E A. Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms[J]. International Microbiology, 2020, 23(1): 5-22.
3	HYNES M J. The Neurospora crassa genome opens up the world of filamentous fungi[J]. Genome Biology, 2003, 4(6): 217.
4	GILES C, LAMONT-FRIEDRICH S J, MICHL T D, et al. The importance of fungal pathogens and antifungal coatings in medical device infections[J]. Biotechnology Advances, 2018, 36(1): 264-280.
5	KUN R S, GOMES A C S, HILDÉN K S, et al. Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation[J]. Biotechnology Advances, 2019, 37(6): 107361.
6	URNOV F D, REBAR E J, HOLMES M C, et al. Genome editing with engineered zinc finger nucleases[J]. Nature Reviews Genetics, 2010, 11(9): 636-646.
7	SHALEM O, SANJANA N E, ZHANG F. High-throughput functional genomics using CRISPR-Cas9[J]. Nature Reviews Genetics, 2015, 16(5): 299-311.
8	BEDELL V M, WANG Y, CAMPBELL J M, et al. In vivo genome editing using a high-efficiency TALEN system[J]. Nature, 2012, 491(7422): 114-118.
9	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.
10	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.
11	BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007, 315(5819): 1709-1712.
12	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.
13	CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823.
14	KOONIN E V, MAKAROVA K S. Origins and evolution of CRISPR-Cas systems[J]. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences, 2019, 374(1772): 20180087.
15	GASIUNAS G, YOUNG J K, KARVELIS T, et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs[J]. Nature Communications, 2020, 11: 5512.
16	HIRANO H, GOOTENBERG J S, HORII T, et al. Structure and engineering of Francisella novicida Cas9[J]. Cell, 2016, 164(5): 950-961.
17	MOJICA F J M, DÍEZ-VILLASEÑOR C, GARCÍA-MARTÍNEZ J, et al. Short motif sequences determine the targets of the prokaryotic CRISPR defence system[J]. Microbiology, 2009, 155(Pt 3): 733-740.
18	HOU Z G, ZHANG Y, PROPSON N E, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis [J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(39): 15644-15649.
19	GASIUNAS G, BARRANGOU R, HORVATH P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39): E2579-E2586.
20	KIM E J, KOO T Y, PARK S W, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni [J]. Nature Communications, 2017, 8: 14500.
21	HIRANO S, ABUDAYYEH O O, GOOTENBERG J S, et al. Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9[J]. Nature Communications, 2019, 10(1): 1968.
22	HARRINGTON L B, PAEZ-ESPINO D, STAAHL B T, et al. A thermostable Cas9 with increased lifetime in human plasma[J]. Nature Communications, 2017, 8(1): 1424.
23	DAS A, HAND T H, SMITH C L, et al. The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9[J]. Nature Communications, 2020, 11(1): 6346.
24	YAMANO T, NISHIMASU H, ZETSCHE B, et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA[J]. Cell, 2016, 165(4): 949-962.
25	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.
26	STRECKER J, JONES S, KOOPAL B, et al. Engineering of CRISPR-Cas12b for human genome editing[J]. Nature Communications, 2019, 10(1): 212.
27	TENG F, CUI T T, FENG G H, et al. Repurposing CRISPR-Cas12b for mammalian genome engineering[J]. Cell Discovery, 2018, 4: 63.
28	KARVELIS T, BIGELYTE G, YOUNG J K, et al. PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage[J]. Nucleic Acids Research, 2020, 48(9): 5016-5023.
29	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.
30	WU Z W, ZHANG Y F, YU H P, et al. Programmed genome editing by a miniature CRISPR-Cas12f nuclease[J]. Nature Chemical Biology, 2021, 17(11): 1132-1138.
31	XIN C C, YIN J H, YUAN S P, et al. Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption[J]. Nature Communications, 2022, 13(1): 5623.
32	LIU J J, ORLOVA N, OAKES B L, et al. CasX enzymes comprise a distinct family of RNA-guided genome editors[J]. Nature, 2019, 566(7743): 218-223.
33	ALTAE-TRAN H, KANNAN S, DEMIRCIOGLU F E, et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases[J]. Science, 2021, 374(6563): 57-65.
34	KARVELIS T, DRUTEIKA G, BIGELYTE G, et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease[J]. Nature, 2021, 599(7886): 692-696.
35	SAITO M, XU P Y, FAURE G, et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease[J]. Nature, 2023, 620(7974): 660-668.
36	PERČULIJA V, LIN J Y, ZHANG B, et al. Functional features and current applications of the RNA-targeting type Ⅵ CRISPR-Cas systems[J]. Advanced Science, 2021, 8(13): 2004685.
37	FONFARA I, RICHTER H, BRATOVIČ M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA[J]. Nature, 2016, 532(7600): 517-521.
38	PAUL B, MONTOYA G. CRISPR-Cas12a: functional overview and applications[J]. Biomedical Journal, 2020, 43(1): 8-17.
39	PICKAR-OLIVER A, GERSBACH C A. The next generation of CRISPR-Cas technologies and applications[J]. Nature Reviews Molecular Cell Biology, 2019, 20(8): 490-507.
40	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.
41	SHEN J Y, ZHAO Q F, HE Q L. Application of CRISPR in filamentous fungi and macrofungi: from component function to development potentiality[J]. ACS Synthetic Biology, 2023, 12(7): 1908-1923.
42	SARKARI P, MARX H, BLUMHOFF M L, et al. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger [J]. Bioresource Technology, 2017, 245(Pt B): 1327-1333.
43	FULLER K K, CHEN S, LOROS J J, et al. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus [J]. Eukaryotic Cell, 2015, 14(11): 1073-1080.
44	LIU Q, ZHANG Y L, LI F Y, et al. Upgrading of efficient and scalable CRISPR-Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila [J]. Biotechnology for Biofuels, 2019, 12: 293.
45	CHEN J J, LAI Y L, WANG L L, et al. CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana [J]. Scientific Reports, 2017, 8: 45763.
46	SHIMA Y, ITO Y, KANEKO S, et al. Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae [J]. Fungal Genetics and Biology, 2009, 46(1): 67-76.
47	FANG Y F, TYLER B M. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9[J]. Molecular Plant Pathology, 2016, 17(1): 127-139.
48	LIU Q, GAO R R, LI J G, et al. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering[J]. Biotechnology for Biofuels, 2017, 10: 1.
49	KATAYAMA T, NAKAMURA H, ZHANG Y, et al. Forced recycling of an AMA1-based genome-editing plasmid allows for efficient multiple gene deletion/integration in the industrial filamentous fungus Aspergillus oryzae [J]. Applied and Environmental Microbiology, 2019, 85(3): e01896-18.
50	CHANG P K. A simple CRISPR/Cas9 system for efficiently targeting genes of Aspergillus section Flavi species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger [J]. Microbiology Spectrum, 2023, 11(1): e0464822.
51	ERDMANN E A, NITSCHE S, GORBUSHINA A A, et al. Genetic engineering of the rock inhabitant Knufia petricola provides insight into the biology of extremotolerant black fungi[J]. Frontiers in Fungal Biology, 2022, 3: 862429.
52	LEISEN T, BIETZ F, WERNER J, et al. CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea [J]. PLoS Pathogens, 2020, 16(8): e1008326.
53	ZHENG X M, ZHENG P, ZHANG K, et al. 5S rRNA promoter for guide RNA expression enabled highly efficient CRISPR/Cas9 genome editing in Aspergillus niger [J]. ACS Synthetic Biology, 2019, 8(7): 1568-1574.
54	YAO G S, CHEN X F, HAN Y J, et al. Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905[J]. Current Genetics, 2022, 68(2): 153-164.
55	NØDVIGC S, NIELSENJ B, KOGLEM E, et al. A CRISPR-Cas9 system for genetic engineering of filamentous fungi[J]. PLoS One, 2015, 10(7): e0133085.
56	ZOU G, XIAO M L, CHAI S X, et al. Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents[J]. Microbial Biotechnology, 2021, 14(6): 2343-2355.
57	SHI T Q, GAO J, WANG W J, et al. CRISPR/Cas9-based genome editing in the filamentous fungus Fusarium fujikuroi and its application in strain engineering for gibberellic acid production[J]. ACS Synthetic Biology, 2019, 8(2): 445-454.
58	HUANG L G, DONG H Z, ZHENG J W, et al. Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion[J]. Microbiological Research, 2019, 223-225: 44-50.
59	CHEN Y Y, CAI C L, YANG J F, et al. Development of the CRISPR-Cas9 system for the marine-derived fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7[J]. Journal of Fungi, 2022, 8(7): 715.
60	ZHANG C, MENG X H, WEI X L, et al. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus [J]. Fungal Genetics and Biology, 2016, 86: 47-57.
61	WEYDA I, YANG L, VANG J, et al. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius [J]. Journal of Microbiological Methods, 2017, 135: 26-34.
62	HAO Z Z, SU X Y. Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex[J]. BMC Biotechnology, 2019, 19(1): 2.
63	GUO M, ZHU X L, LI H X, et al. Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae [J]. AMB Express, 2016, 6(1): 57.
64	YANG J F, ZHOU Z B, CHEN Y Y, et al. Characterization of the depsidone gene cluster reveals etherification, decarboxylation and multiple halogenations as tailoring steps in depsidone assembly[J]. Acta Pharmaceutica Sinica B, 2023, 13(9): 3919-3929.
65	PERFEITO L, FERNANDES L, MOTA C, et al. Adaptive mutations in bacteria: high rate and small effects[J]. Science, 2007, 317(5839): 813-815.
66	ROACH K C, HEITMAN J. Unisexual reproduction reverses Muller’s ratchet[J]. Genetics, 2014, 198(3): 1059-1069.
67	BOEKE J D, LACROUTE F, FINK G R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance[J]. Molecular & General Genetics: MGG, 1984, 197(2): 345-346.
68	DONG H Z, ZHENG J W, YU D, et al. Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes[J]. Journal of Microbiological Methods, 2019, 163: 105655.
69	NØDVIG C S, HOOF J B, KOGLE M E, et al. Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli[J]. Fungal Genetics and Biology, 2018, 115: 78-89.
70	CHENY Y, YANGJ F, CAIC L, et al. Development of marker recycling systems for sequential genetic manipulation in marine-derived fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7[J]. Journal of Fungi, 2023, 9(3): 302.
71	FOSTER A J, MARTIN-URDIROZ M, YAN X, et al. CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus[J]. Scientific Reports, 2018, 8(1): 14355.
72	HANDELMAN M, OSHEROV N. Efficient generation of multiple seamless point mutations conferring triazole resistance in Aspergillus fumigatus [J]. Journal of Fungi, 2023, 9(6): 644.
73	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.
74	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.
75	WANG Q, COBINE P A, COLEMAN J J. Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes[J]. Fungal Genetics and Biology, 2018, 117: 21-29.
76	DAVIS K A, SAMPSON J K, PANACCIONE D G. Genetic reprogramming of the ergot alkaloid pathway of Metarhizium brunneum [J]. Applied and Environmental Microbiology, 2020, 86(19): e01251-20.
77	HICKS C, WITTE T E, SPROULE A, et al. CRISPR-Cas9 gene editing and secondary metabolite screening confirm Fusarium graminearum C16 biosynthetic gene cluster products as decalin-containing diterpenoid pyrones[J]. Journal of Fungi, 2023, 9(7): 695.
78	LI S J, SONG Z Y, LIU C Y, et al. Biomimetic mineralization-based CRISPR/Cas9 ribonucleoprotein nanoparticles for gene editing[J]. ACS Applied Materials & Interfaces, 2019, 11(51): 47762-47770.
79	CAIRNS T C, ZHENG X M, FEURSTEIN C, et al. A library of Aspergillus niger chassis strains for morphology engineering connects strain fitness and filamentous growth with submerged macromorphology[J]. Frontiers in Bioengineering and Biotechnology, 2021, 9: 820088.
80	GUEGAN H, POIRIER W, RAVENEL K, et al. Deciphering the role of PIG1 and DHN-melanin in Scedosporium apiospermum conidia[J]. Journal of Fungi, 2023, 9(2): 134.
81	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.
82	JIANG C M, LV G B, TU Y Y, et al. Applications of CRISPR/Cas9 in the synthesis of secondary metabolites in filamentous fungi[J]. Frontiers in Microbiology, 2021, 12: 638096.
83	DONG L B, LIN X T, YU D, et al. High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation[J]. Journal of Industrial Microbiology & Biotechnology, 2020, 47(1): 133-144.
84	ZHENG Y M, LIN F L, GAO H, et al. Development of a versatile and conventional technique for gene disruption in filamentous fungi based on CRISPR-Cas9 technology[J]. Scientific Reports, 2017, 7(1): 9250.
85	WEI T Y, WU Y J, XIE Q P, et al. CRISPR/Cas9-based genome editing in the filamentous fungus Glarea lozoyensis and its application in manipulating gloF [J]. ACS Synthetic Biology, 2020, 9(8): 1968-1977.
86	XIANG B Y, HAO X R, XIE Q H, et al. Deletion of a rare fungal PKS CgPKS11 promotes chaetoglobosin A biosynthesis, yet defers the growth and development of Chaetomium globosum [J]. Journal of Fungi, 2021, 7(9): 750.
87	ÖKMEN B, SCHWAMMBACH D, BAKKEREN G, et al. The Ustilago hordei-barley interaction is a versatile system for characterization of fungal effectors[J]. Journal of Fungi, 2021, 7(2): 86.
88	SONG L T, OUEDRAOGO J P, KOLBUSZ M, et al. Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger [J]. PLoS One, 2018, 13(8): e0202868.
89	DENG H X, GAO R J, LIAO X R, et al. Genome editing in Shiraia bambusicola using CRISPR-Cas9 system[J]. Journal of Biotechnology, 2017, 259: 228-234.
90	MÓZSIK L, HOEKZEMA M, DE KOK N A W, et al. CRISPR-based transcriptional activation tool for silent genes in filamentous fungi[J]. Scientific Reports, 2021, 11(1): 1118.
91	BALDIN C, KÜHBACHER A, MERSCHAK P, et al. Inducible selectable marker genes to improve Aspergillus fumigatus genetic manipulation[J]. Journal of Fungi, 2021, 7(7): 506.
92	WEBER J, VALIANTE V, NØDVIG C S, et al. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing[J]. ACS Synthetic Biology, 2017, 6(1): 62-68.
93	KUN R S, MENG J L, SALAZAR-CEREZO S, et al. CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass[J]. Enzyme and Microbial Technology, 2020, 136: 109508.
94	WANG Q, ZHAO Q Q, LIU Q, et al. CRISPR/Cas9-mediated genome editing in Penicillium oxalicum and Trichoderma reesei using 5S rRNA promoter-driven guide RNAs[J]. Biotechnology Letters, 2021, 43(2): 495-502.
95	LI J Y, WANG X, ZOU J H, et al. Identification and characterization of the determinants of copper resistance in the acidophilic fungus Acidomyces richmondensis MEY-1 using the CRISPR/Cas9 system[J]. Applied and Environmental Microbiology, 2023, 89(3): e0210722.
96	JARCZYNSKA Z D, GARCIA VANEGAS K, DEICHMANN M, et al. A versatile in vivo DNA assembly toolbox for fungal strain engineering[J]. ACS Synthetic Biology, 2022, 11(10): 3251-3263.
97	POHL C, MÓZSIK L, DRIESSEN A J M, et al. Genome editing in Penicillium chrysogenum using Cas9 ribonucleoprotein particles[J]. Methods in Molecular Biology, 2018, 1772: 213-232.
98	CHEN Z Q, ZHANG C, PEI L L, et al. Production of L-malic acid by metabolically engineered Aspergillus nidulans based on efficient CRISPR-Cas9 and cre-loxP systems[J]. Journal of Fungi, 2023, 9(7): 719.
99	于鲲, 薛佳琪, 王进宽, 等. CRISPR/Cas9基因编辑技术在丝状真菌中的应用[J]. 生物技术进展, 2022, 12(5): 696-704.
	YU K, XUE J Q, WANG J K, et al. Research progress on application of CRISPR/Cas9 gene editing technique in filamentous fungi[J]. Current Biotechnology, 2022, 12(5): 696-704.
100	LU S, SHEN X R, CHEN B S. Development of an efficient vector system for gene knock-out and near in-cis gene complementation in the sugarcane smut fungus[J]. Scientific Reports, 2017, 7(1): 3113.
101	VIEIRA A A, VIANNA G R, CARRIJO J, et al. Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system[J]. Scientific Reports, 2021, 11(1): 1085.
102	BRUNI G O, ZHONG K, LEE S C, et al. CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar [J]. Fungal Genetics and Biology, 2019, 124: 1-7.
103	LI D D, TANG Y, LIN J, et al. Methods for genetic transformation of filamentous fungi[J]. Microbial Cell Factories, 2017, 16(1): 168.
104	STROHKENDL I, SAIFUDDIN F A, RYBARSKI J R, et al. Kinetic basis for DNA target specificity of CRISPR-Cas12a[J]. Molecular Cell, 2018, 71(5): 816-824.e3.
105	CAMPA C C, WEISBACH N R, SANTINHA A J, et al. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts[J]. Nature Methods, 2019, 16(9): 887-893.
106	JIMÉNEZ A, HOFF B, REVUELTA J L. Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1[J]. New Biotechnology, 2020, 57: 29-33.
107	LI S Y, CHENG Q X, WANG J M, et al. CRISPR-Cas12a-assisted nucleic acid detection[J]. Cell Discovery, 2018, 4: 20.
108	MU K Q, REN X J, YANG H, et al. CRISPR-Cas12a-based diagnostics of wheat fungal diseases[J]. Journal of Agricultural and Food Chemistry, 2022, 70(23): 7240-7247.
109	HUANG J, ROWE D, SUBEDI P, et al. CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae [J]. Nature Communications, 2022, 13(1): 7168.
110	HUANG J, COOK D E. CRISPR-Cas12a ribonucleoprotein-mediated gene editing in the plant pathogenic fungus Magnaporthe oryzae [J]. STAR Protocols, 2022, 3(1): 101072.
111	ABDULRACHMAN D, EURWILAICHITR L, CHAMPREDA V, et al. Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277[J]. BMC Biotechnology, 2021, 21(1): 15.
112	VANEGAS K G, JARCZYNSKA Z D, STRUCKO T, et al. Cpf1 enables fast and efficient genome editing in Aspergilli[J]. Fungal Biology and Biotechnology, 2019, 6: 6.
113	KWON M J, SCHÜTZE T, SPOHNER S, et al. Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi[J]. Fungal Biology and Biotechnology, 2019, 6: 15.
114	KATAYAMA T, MARUYAMA J I. CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae [J]. Journal of Bioscience and Bioengineering, 2022, 133(4): 353-361.
115	CHEN T M, CHEN Z M, ZHANG H X, et al. Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae [J]. Folia Microbiologica, 2024, 69: 373-382.
116	KOMOR A C, KIM Y B, PACKER M S, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424.
117	PORTO E M, KOMOR A C, SLAYMAKER I M, et al. Base editing: advances and therapeutic opportunities[J]. Nature Reviews Drug Discovery, 2020, 19(12): 839-859.
118	KOMOR A C, ZHAO K T, PACKER M S, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: a base editors with higher efficiency and product purity[J]. Science Advances, 2017, 3(8): eaao4774.
119	王雁南, 孙宇辉. 碱基编辑技术及其在微生物合成生物学中的应用[J]. 合成生物学, 2023, 4(4): 720-737.
	WANG Y N, SUN Y H. Base editing technology and its application in microbial synthetic biology[J]. Synthetic Biology Journal, 2023,4(4): 720-737.
120	UMEYAMA T, HAYASHI Y, SHIMOSAKA H, et al. CRISPR/Cas9 genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus [J]. Antimicrobial Agents and Chemotherapy, 2018, 62(9): e00894-18.
121	KIM D S, LIM K Y, KIM S T, et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases[J]. Nature Biotechnology, 2017, 35(5): 475-480.
122	LAHR W S, SIPE C J, SKEATE J G, et al. CRISPR-Cas9 base editors and their current role in human therapeutics[J]. Cytotherapy, 2023, 25(3): 270-276.
123	ZHANG C Y, LI N, RAO L, et al. Development of an efficient C-to-T base-editing system and its application to cellulase transcription factor precise engineering in thermophilic fungus Myceliophthora thermophila [J]. Microbiology Spectrum, 2022, 10(3): e0232121.
124	ZHAO F L, SUN C X, LIU Z W, et al. Multiplex base-editing enables combinatorial epigenetic regulation for genome mining of fungal natural products[J]. Journal of the American Chemical Society, 2023, 145(1): 413-421.
125	WANG J Y, DOUDNA J A. CRISPR technology: a decade of genome editing is only the beginning[J]. Science, 2023, 379(6629): eadd8643.
126	MÓZSIK L, POHL C, MEYER V, et al. Modular synthetic biology toolkit for filamentous fungi[J]. ACS Synthetic Biology, 2021, 10(11): 2850-2861.
127	CHAVEZ A, SCHEIMAN J, VORA S, et al. Highly efficient Cas9-mediated transcriptional programming[J]. Nature Methods, 2015, 12(4): 326-328.
128	GILBERT L A, LARSON M H, MORSUT L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154(2): 442-451.
129	CHEN M, QI L S. Repurposing CRISPR system for transcriptional activation[J]. Advances in Experimental Medicine and Biology, 2017, 983: 147-157.
130	ROUX I, WOODCRAFT C, HU J Y, et al. CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi[J]. ACS Synthetic Biology, 2020, 9(7): 1843-1854.
131	DOMINGUEZ A A, LIM W A, QI L S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation[J]. Nature Reviews Molecular Cell Biology, 2016, 17(1): 5-15.
132	GHAVAMI S, PANDI A. CRISPR interference and its applications[J]. Progress in Molecular Biology and Translational Science, 2021, 180: 123-140.
133	LARSON M H, GILBERT L A, WANG X W, et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression[J]. Nature Protocols, 2013, 8(11): 2180-2196.
134	ZHANG Y M, ZHENG L, XIE K B. CRISPR/dCas9-mediated gene silencing in two plant fungal pathogens[J]. mSphere, 2023, 8(1): e0059422.
135	NAKAMURA M, GAO Y C, DOMINGUEZ A A, et al. CRISPR technologies for precise epigenome editing[J]. Nature Cell Biology, 2021, 23(1): 11-22.
136	PULECIO J, VERMA N, MEJÍA-RAMÍREZ E, et al. CRISPR/Cas9-based engineering of the epigenome[J]. Cell Stem Cell, 2017, 21(4): 431-447.
137	HILTON I B, D’IPPOLITO A M, VOCKLEY C M, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[J]. Nature Biotechnology, 2015, 33(5): 510-517.
138	LI X J, HUANG L G, PAN L J, et al. CRISPR/dCas9-mediated epigenetic modification reveals differential regulation of histone acetylation on Aspergillus niger secondary metabolite[J]. Microbiological Research, 2021, 245: 126694.
139	NUÑEZ J K, CHEN J, POMMIER G C, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing[J]. Cell, 2021, 184(9): 2503-2519.e17.
140	CHEN X Y, MORAN TORRES J P, LI Y L, et al. Inheritable CRISPR based epigenetic modification in a fungus[J]. Microbiological Research, 2023, 272: 127397.
141	LI X Y, AWAKAWA T, MORI T, et al. Heterodimeric non-heme iron enzymes in fungal meroterpenoid biosynthesis[J]. Journal of the American Chemical Society, 2021, 143(50): 21425-21432.
142	YE W, LIU T M, LIU Y P, et al. Enhancing gliotoxins production in deep-sea derived fungus Dichotomocyes cejpii by engineering the biosynthetic pathway[J]. Bioresource Technology, 2023, 377: 128905.
143	WANG D D, JIN S D, LU Q H, et al. Advances and challenges in CRISPR/Cas-based fungal genome engineering for secondary metabolite production: a review[J]. Journal of Fungi, 2023, 9(3): 362.
144	XIAO A, CHENG Z C, KONG L, et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool[J]. Bioinformatics, 2014, 30(8): 1180-1182.
145	LABUN K, MONTAGUE T G, GAGNON J A, et al. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering[J]. Nucleic Acids Research, 2016, 44(W1): W272-W276.
146	LEE J K, JEONG E, LEE J, et al. Directed evolution of CRISPR-Cas9 to increase its specificity[J]. Nature Communications, 2018, 9(1): 3048.
147	RABINOWITZ R, OFFEN D. Single-base resolution: increasing the specificity of the CRISPR-Cas system in gene editing[J]. Molecular Therapy, 2021, 29(3): 937-948.

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	约3 bp 5′ of PAM	[12]
FnCas9	1629	NGG	20 bp	约3 bp 5′ of PAM	[16]
SaCas9	1053	NNGR RT	21 bp	约3 bp 5′ of PAM	[17]
NmCas9	1082	NNNNG ATT	24 bp	约3 bp 5′ of PAM	[18]
St1Cas9	1121	NNAGA AW	20 bp	约3 bp 5′ of PAM	[13,19]
St3Cas9	1409	NGGNG	20 bp	约3 bp 5′ of PAM	[13,19]
CjCas9	984	NNNNACAC	22 bp	约3 bp 5′ of PAM	[20]
CdCas9	1084	NNRHHHY	22 bp	约3 bp 5′ of PAM	[21]
GeoCas9	1087	NNNNCRAA	21/22 bp	约3 bp 5′ of PAM	[22]
AceCas9	1138	NNNCC	20 bp	约3 bp 5′ of PAM	[23]
AsCas12a	1307	TTTV	24 bp	约19/24 bp 3′ of PAM	[24]
LbCas12a	1228	TTTV	24 bp	约19/24 bp 3′ of PAM	[24]
FnCas12a	1307	TTV	24 bp	约19/24 bp 3′ of PAM	[25]
Cas12b	1108/1130	TTN	23 bp	约17/23 bp 5′ of PAM	[26-27]
Cas12f	约400～600	5′ T/C-rich	20 bp/33～39 bp	约3/24 bp 3′ of PAM	[28-31]
CasX	978	TTCN	20 bp	约12/25 bp ′ of 3′ PAM	[32]
TnpB/IscB/IsrB	约400	TTGAT/ATAAA /ATGA/NNG	15～45 bp	约3/12 bp of 5′ TAM 约6/21 bp of 3′ TAM	[33-34]
Fz	约500～800	CATA/TTAAN /CCG/TAG	7～21 bp	约9/21 bp of 5′ TAM	[35]
Cas14	约400～700	ssDNA	20 bp		[29]
Cas13	约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	约3 bp 5′ of PAM	[12]
FnCas9	1629	NGG	20 bp	约3 bp 5′ of PAM	[16]
SaCas9	1053	NNGR RT	21 bp	约3 bp 5′ of PAM	[17]
NmCas9	1082	NNNNG ATT	24 bp	约3 bp 5′ of PAM	[18]
St1Cas9	1121	NNAGA AW	20 bp	约3 bp 5′ of PAM	[13,19]
St3Cas9	1409	NGGNG	20 bp	约3 bp 5′ of PAM	[13,19]
CjCas9	984	NNNNACAC	22 bp	约3 bp 5′ of PAM	[20]
CdCas9	1084	NNRHHHY	22 bp	约3 bp 5′ of PAM	[21]
GeoCas9	1087	NNNNCRAA	21/22 bp	约3 bp 5′ of PAM	[22]
AceCas9	1138	NNNCC	20 bp	约3 bp 5′ of PAM	[23]
AsCas12a	1307	TTTV	24 bp	约19/24 bp 3′ of PAM	[24]
LbCas12a	1228	TTTV	24 bp	约19/24 bp 3′ of PAM	[24]
FnCas12a	1307	TTV	24 bp	约19/24 bp 3′ of PAM	[25]
Cas12b	1108/1130	TTN	23 bp	约17/23 bp 5′ of PAM	[26-27]
Cas12f	约400～600	5′ T/C-rich	20 bp/33～39 bp	约3/24 bp 3′ of PAM	[28-31]
CasX	978	TTCN	20 bp	约12/25 bp ′ of 3′ PAM	[32]
TnpB/IscB/IsrB	约400	TTGAT/ATAAA /ATGA/NNG	15～45 bp	约3/12 bp of 5′ TAM 约6/21 bp of 3′ TAM	[33-34]
Fz	约500～800	CATA/TTAAN /CCG/TAG	7～21 bp	约9/21 bp of 5′ TAM	[35]
Cas14	约400～700	ssDNA	20 bp		[29]
Cas13	约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]等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	Myceliophthora thermophila^[44], Beauveria bassiana^[45]等
	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]等
	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	Phytophthora sojae^[47], M. thermophila^[48]等
	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]等
	Chlorimuron (sur)	Sur (乙酰乳酸合成酶)具有氯嘧磺隆抗性	Knufia petricola^[51]等
	Fenhexamid (Fen^r)	点突变的 ERG27 (编码酮还原酶),具有环酰菌胺抗性	Botrytis cinerea^[52]等
	Nourseothricin (Nat)	Nat (编码N-乙酰转移酶),具有诺尔斯菌素抗性	B. cinerea^[52]等
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]等
	niaD	niaD功能缺陷突变株能够耐受高浓度氯盐	A. oryzae^[49]等
	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]等
	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]等
	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]等
	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]等
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]等
	wA	wA突变体形成白色分生孢子	A. oryzae^[49],A. nidulans^[55]等
	yA	yA突变体形成黄色分生孢子	A. oryzae^[49], A.nidulans^[55]等
	fwnA	fwnA突变体形成白色分生孢子	A. niger^[58]等
	albA	albA突变体形成白色分生孢子	A. niger^[53]等
	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大大减小	A. niger^[53]等
	creA	creA 敲除菌株产孢能力显著降低	Spiromastix sp. ^[59]等
	pksP	pksP敲除菌落具有明显的附白化表型	A. fumigatus^[60]等
	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]等
	ayg1	ayg1 敲除可导致孢子的颜色从黑色变为黄色	A. carbonarius^[61]等
	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	Trichoderma reesei^[62]等

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]等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	Myceliophthora thermophila^[44], Beauveria bassiana^[45]等
	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]等
	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	Phytophthora sojae^[47], M. thermophila^[48]等
	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]等
	Chlorimuron (sur)	Sur (乙酰乳酸合成酶)具有氯嘧磺隆抗性	Knufia petricola^[51]等
	Fenhexamid (Fen^r)	点突变的 ERG27 (编码酮还原酶),具有环酰菌胺抗性	Botrytis cinerea^[52]等
	Nourseothricin (Nat)	Nat (编码N-乙酰转移酶),具有诺尔斯菌素抗性	B. cinerea^[52]等
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]等
	niaD	niaD功能缺陷突变株能够耐受高浓度氯盐	A. oryzae^[49]等
	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]等
	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]等
	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]等
	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]等
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]等
	wA	wA突变体形成白色分生孢子	A. oryzae^[49],A. nidulans^[55]等
	yA	yA突变体形成黄色分生孢子	A. oryzae^[49], A.nidulans^[55]等
	fwnA	fwnA突变体形成白色分生孢子	A. niger^[58]等
	albA	albA突变体形成白色分生孢子	A. niger^[53]等
	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大大减小	A. niger^[53]等
	creA	creA 敲除菌株产孢能力显著降低	Spiromastix sp. ^[59]等
	pksP	pksP敲除菌落具有明显的附白化表型	A. fumigatus^[60]等
	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]等
	ayg1	ayg1 敲除可导致孢子的颜色从黑色变为黄色	A. carbonarius^[61]等
	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	Trichoderma reesei^[62]等

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]等
	PtrpC	吲哚甘油磷酸合成酶启动子	N. crassa^[81], Siliqua minima^[84]等
	PgpdA	3-磷酸甘油醛脱氢酶的启动子	F. fujikuroi^[57], A. fumigatus^[60]等
	Ppdc	丙酮酸脱羧酶的启动子	T. reesei^[40], Glarea lozoyensis^[85]等
	Pactin	肌动蛋白的启动子	Chaetomium globosum^[86]Leptosphaeria maculans^[85]等
	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	Shiraia 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]等
	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]等
	niiA	硝酸还原酶的启动子	A. fumigatus^[60]等
RNA聚合酶Ⅲ型启动子	u6	人U6微核启动子	A.oryzae^[49], A. richmondensis^[95]等
	5S rRNA	5S rRNA基因启动子	A. niger^[53], P. oxalicum^[94]等
	tRNA	转录转移核糖核酸启动子	A. niger^[93], A. aculeatus^[96]等
	SNR52	核仁小分子RNA 52启动子	N. crassa^[81], A. fumigatus^[43]等
体外转录	T7	T7噬菌体衍生启动子	T. reesei^[62], P. chrysogenum^[97]等

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

CRISPR/Cas systems and their applications in gene editing with 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%～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有显著不同（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%～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；PgpdA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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[2]	许志锰, 谢震. 引导编辑研究进展及其应用[J]. 合成生物学, 2024, 5(1): 1-15.
[3]	陈雅如, 曹英秀, 宋浩. 电活性微生物基因编辑与转录调控技术进展与应用[J]. 合成生物学, 2023, 4(6): 1281-1299.
[4]	林继聪, 邹根, 刘宏民, 魏勇军. CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用[J]. 合成生物学, 2023, 4(4): 738-755.
[5]	柳柯, 林桂虹, 刘坤, 周伟, 王风清, 魏东芝. CRISPR/Cas系统的挖掘、改造与功能拓展[J]. 合成生物学, 2023, 4(1): 47-66.
[6]	滕小龙, 史硕博. CRISPR/Cas9系统在基因组编辑中的优化与发展[J]. 合成生物学, 2023, 4(1): 67-85.
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