Research Progress in CRISPR/Cas9 Genome Editing System in Edible and Medicinal Fungi

doi:10.13560/j.cnki.biotech.bull.1985.2021-1055

Abstract

Abstract:

CRISPR/Cas9 is currently the most successful precise genome editing technology. So far,there have been relevant application reports in at least nine edible and medicinal fungi,including Pleurotus eryngii,Pleurotus ostreatus,Agaricus bisporus,Ganoderma lucidum,Schizophyllum commune,Coprinopsis cinerea,Flammulina velutipes,Cordyceps militaris and Shiraia bambusicola. This article briefly describes the mechanism and delivery strategies of the CRISPR/Cas9 system,and proposes solutions for the application in edible and medicinal fungi. Last but not least,referring to the current frontier development of CRISPR technology,such as multiplex gene editing, base editing, prime editing and transcription regulation, etc., we give certain enlightenment to the future application prospects in the field of edible and medicinal fungi.

Key words: CRISPR/Cas9, edible and medicinal fungi, genome editing, RNP

LIU Xiao-tian, QIU Hao, TIAN Li, REN Ang, ZHAO Ming-wen. Research Progress in CRISPR/Cas9 Genome Editing System in Edible and Medicinal Fungi[J]. Biotechnology Bulletin, 2021, 37(11): 4-13.

Figures/Tables 2

References 67

[1]	Liu YN, Lu XX, Chen D, et al. Phospholipase D and phosphatidic acid mediate heat stress induced secondary metabolism in Ganoderma lucidum[J]. Environ Microbiol, 2017, 19(11): 4657-4669. doi: 10.1111/emi.2017.19.issue-11 URL
[2]	Li L, Chang SS, Liu Y. RNA interference pathways in filamentous fungi[J]. Cell Mol Life Sci, 2010, 67(22): 3849-3863. doi: 10.1007/s00018-010-0471-y URL
[3]	Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes[J]. Nat Biotechnol, 2014, 32(4): 347-355. doi: 10.1038/nbt.2842 URL
[4]	Adli M. The CRISPR tool kit for genome editing and beyond[J]. Nat Commun, 2018, 9(1): 1911. doi: 10.1038/s41467-018-04252-2 URL
[5]	Liu R, Chen L, Jiang Y, et al. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system[J]. Cell Discov, 2015, 1: 15007. doi: 10.1038/celldisc.2015.7 URL
[6]	Kim S, Kim D, Cho SW, et al. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins[J]. Genome Res, 2014, 24(6): 1012-1019. doi: 10.1101/gr.171322.113 URL
[7]	DiCarlo JE, Norville JE, Mali P, et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J]. Nucleic Acids Res, 2013, 41(7): 4336-4343. doi: 10.1093/nar/gkt135 pmid: 23460208
[8]	Zhang C, Meng X, Wei X, et al. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus[J]. Fungal Genet Biol, 2016, 86: 47-57. doi: S1087-1845(15)30055-4 pmid: 26701308
[9]	Wang T, Yue S, Jin Y, et al. Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii[J]. Fungal Genet Biol, 2021, 147: 103509. doi: 10.1016/j.fgb.2020.103509 URL
[10]	Boontawon T, Nakazawa T, Xu HB, et al. Gene targeting using pre-assembled Cas9 ribonucleoprotein and split-marker recombination in Pleurotus ostreatus[J]. FEMS Microbiol Lett, 2021, 368(13): fnab080. doi: 10.1093/femsle/fnab080 URL
[11]	Boontawon T, Nakazawa T, Inoue C, et al. Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus[J]. AMB Express, 2021, 11(1): 30. doi: 10.1186/s13568-021-01193-w pmid: 33609205
[12]	Boontawon T, Nakazawa T, Horii M, et al. Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9[J]. Fungal Genet Biol, 2021, 154: 103599. doi: 10.1016/j.fgb.2021.103599 pmid: 34153439
[13]	吕阳, 贺晓飞, 王佩, 等. 基于CRISPR系统的双孢蘑菇基因编辑体系建立及应用[J]. 食用菌学报, 2020, 27(3): 16-22.
	Lv Y, He XF, Wang P, et al. Establishment of a CRISPR/Cas9 system in Agaricus bisporus[J]. Acta Edulis Fungi, 2020, 27(3): 16-22.
[14]	Liu K, Sun B, You H, et al. Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system[J]. Microb Biotechnol, 2020, 13(2): 386-396. doi: 10.1111/1751-7915.13534 pmid: 31958883
[15]	Wang PA, Xiao H, Zhong JJ. CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum[J]. Appl Microbiol Biotechnol, 2020, 104(4): 1661-1671. doi: 10.1007/s00253-019-10298-z URL
[16]	Qin H, Xiao H, Zou G, et al. CRISPR-Cas9 assisted gene disruption in the higher fungus Ganoderma species[J]. Process Biochem, 2017, 56: 57-61. doi: 10.1016/j.procbio.2017.02.012 URL
[17]	Jan Vonk P, Escobar N, Wösten HAB, et al. High-throughput targeted gene deletion in the model mushroom Schizophyllum commune using pre-assembled Cas9 ribonucleoproteins[J]. Sci Rep, 2019, 9(1): 7632. doi: 10.1038/s41598-019-44133-2 URL
[18]	Sugano SS, Suzuki H, Shimokita E, et al. Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system[J]. Sci Rep, 2017, 7(1): 1260. doi: 10.1038/s41598-017-00883-5 URL
[19]	刘建雨, 刘建辉, 张丹, 等. 农杆菌介导的Cas9基因转化金针菇的研究[J]. 食用菌学报, 2017, 24(3): 25-29.
	Liu JY, Liu JH, Zhang D, et al. Agrobacterium-mediated gene transformation of Cas9 into Flammulina velutipes[J]. Acta Edulis Fungi, 2017, 24(3): 25-29.
[20]	林金德, 杨雪琴, 魏韬, 等. 金针菇G蛋白偶联受体基因的CRISPR/Cas9基因组编辑载体构建及转化研究[J]. 菌物学报, 2019, 38(3): 349-361.
	Lin JD, Yang XQ, Wei T, et al. Construction and transformation of CRISPR/Cas9 genome editing vector of Flammulina filiformis G protein-coupled receptor gene[J]. Mycosystema, 2019, 38(3): 349-361.
[21]	欧阳萍兰, 李琼洁, 郭丽琼, 等. 基于CRISPR/Cas9技术研究金针菇冷诱导结实基因HK1和HK2的编辑转化系统[J]. 食用菌学报, 2018, 25(3): 1-7, 113.
	Ouyang PL, Li QJ, Guo LQ, et al. Establishment of a CRISPR/Cas9 system for editing cold-induced gene HK₁/HK₂ in Flammulina velutipes[J]. Acta Edulis Fungi, 2018, 25(3): 1-7, 113.
[22]	Chen BX, Wei T, Ye ZW, et al. Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris[J]. Front Microbiol, 2018, 9: 1157. doi: 10.3389/fmicb.2018.01157 URL
[23]	Deng H, Gao R, Liao X, et al. Genome editing in Shiraia bambusicola using CRISPR-Cas9 system[J]. J Biotechnol, 2017, 259: 228-234. doi: 10.1016/j.jbiotec.2017.06.1204 URL
[24]	Waltz E. Gene-edited CRISPR mushroom escapes US regulation[J]. Nature, 2016, 532(7599): 293. doi: 10.1038/nature.2016.19754 URL
[25]	Zou G, Xiao M, Chai S, et al. Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents[J]. Microb Biotechnol, 2020. DOI: 10.1111/1751-7915.13652. doi: 10.1111/1751-7915.13652
[26]	Kunitake E, Tanaka T, Ueda H, et al. CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica[J]. Fungal Genet Biol, 2019, 130: 82-90. doi: S1087-1845(18)30251-2 pmid: 31026589
[27]	Liu W, An C, Shu X, et al. A dual-plasmid CRISPR/cas system for mycotoxin elimination in polykaryotic industrial fungi[J]. ACS Synth Biol, 2020, 9(8): 2087-2095. doi: 10.1021/acssynbio.0c00178 URL
[28]	Li C, Zong Y, Wang Y, et al. Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion[J]. Genome Biol, 2018, 19(1): 59. doi: 10.1186/s13059-018-1443-z URL
[29]	Métais JY, Doerfler PA, Mayuranathan T, et al. Genome editing of HBG1 and HBG2 to induce fetal hemoglobin[J]. Blood Adv, 2019, 3(21): 3379-3392. doi: 10.1182/bloodadvances.2019000820 URL
[30]	Gao Y, Zhao Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing[J]. J Integr Plant Biol, 2014, 56(4): 343-349. doi: 10.1111/jipb.v56.4 URL
[31]	He Y, Zhang T, Yang N, et al. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing[J]. J Genet Genomics, 2017, 44(9): 469-472. doi: 10.1016/j.jgg.2017.08.003 URL
[32]	Ishi K, Maruyama J, Juvvadi PR, et al. Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae:multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae[J]. Biosci Biotechnol Biochem, 2005, 69(4): 747-754. doi: 10.1271/bbb.69.747 URL
[33]	Hara S, Jin FJ, Takahashi T, et al. A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae[J]. Mol Genet Genomics, 2012, 287(2): 177-187. doi: 10.1007/s00438-011-0669-1 URL
[34]	Chen J, Lai Y, Wang L, et al. CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana[J]. Sci Rep, 2017, 8: 45763.
[35]	Broomfield PL, Hargreaves JA. A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis[J]. Curr Genet, 1992, 22(2): 117-121. pmid: 1423716
[36]	Irie T, Sato T, Saito K, et al. Construction of a homologous selectable marker gene for Lentinula edodes transformation[J]. Biosci Biotechnol Biochem, 2003, 67(9): 2006-2009. doi: 10.1271/bbb.67.2006 URL
[37]	Li H, Zhong JJ. Role of calcineurin-responsive transcription factor CRZ1 in ganoderic acid biosynjournal by Ganoderma lucidum[J]. Process Biochem, 2020, 95: 166-173. doi: 10.1016/j.procbio.2020.05.027 URL
[38]	Gritz L, Davies J. Plasmid-encoded hygromycin B resistance:the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae[J]. Gene, 1983, 25(2/3): 179-188. doi: 10.1016/0378-1119(83)90223-8 URL
[39]	Hu Y, Xu W, Hu S, et al. In Ganoderma lucidum, Glsnf1 regulates cellulose degradation by inhibiting GlCreA during the utilization of cellulose[J]. Environ Microbiol, 2020, 22(1): 107-121. doi: 10.1111/emi.v22.1 URL
[40]	Thompson CJ, Movva NR, Tizard R, et al. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus[J]. EMBO J, 1987, 6(9): 2519-2523. pmid: 16453790
[41]	Miao Y, Xia Y, Kong Y, et al. Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi[J]. Environ Microbiol, 2021, 23(6): 2937-2954. doi: 10.1111/emi.v23.6 URL
[42]	Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J]. Nat Biotechnol, 2013, 31(9): 822-826. doi: 10.1038/nbt.2623 URL
[43]	Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases[J]. Nat Biotechnol, 2013, 31(9): 827-832. doi: 10.1038/nbt.2647 URL
[44]	Schuster M, Schweizer G, Reissmann S, et al. Genome editing in Ustilago maydis using the CRISPR-Cas system[J]. Fungal Genet Biol, 2016, 89: 3-9. doi: S1087-1845(15)30025-6 pmid: 26365384
[45]	Slaymaker IM, Gao L, Zetsche B, et al. Rationally engineered Cas9 nucleases with improved specificity[J]. Science, 2016, 351(6268): 84-88. doi: 10.1126/science.aad5227 pmid: 26628643
[46]	Kleinstiver BP, Pattanayak V, Prew MS, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects[J]. Nature, 2016, 529(7587): 490-495. doi: 10.1038/nature16526 URL
[47]	Tan YY, Chu AHY, Bao SY, et al. Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity[J]. PNAS, 2019, 116(42): 20969-20976. doi: 10.1073/pnas.1906843116 URL
[48]	Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6121): 823-826. doi: 10.1126/science.1232033 URL
[49]	Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424. doi: 10.1038/nature17946 URL
[50]	Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681): 464-471. doi: 10.1038/nature24644 URL
[51]	Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 2019, 576(7785): 149-157. doi: 10.1038/s41586-019-1711-4 URL
[52]	Chavez A, Scheiman J, Vora S, et al. Highly efficient Cas9-mediated transcriptional programming[J]. Nat Methods, 2015, 12(4): 326-328. doi: 10.1038/nmeth.3312 URL
[53]	Gilbert LA, Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154(2): 442-451. doi: 10.1016/j.cell.2013.06.044 pmid: 23849981
[54]	Nishimasu H, Shi X, Ishiguro S, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space[J]. Science, 2018, 361(6408): 1259-1262. doi: 10.1126/science.aas9129 pmid: 30166441
[55]	Kleinstiver BP, Prew MS, Tsai SQ, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 2015, 523(7561): 481-485. doi: 10.1038/nature14592 URL
[56]	Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3): 759-771. doi: 10.1016/j.cell.2015.09.038 pmid: 26422227
[57]	Fagerlund RD, Staals RH, Fineran PC. The Cpf1 CRISPR-Cas protein expands genome-editing tools[J]. Genome Biol, 2015, 16: 251. doi: 10.1186/s13059-015-0824-9 pmid: 26578176
[58]	Gao Z, Fan M, Das AT, et al. Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA[J]. Nucleic Acids Res, 2020, 48(10): 5527-5539. doi: 10.1093/nar/gkaa226 URL
[59]	Zong Y, Wang YP, Li C, et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nat Biotechnol, 2017, 35(5): 438-440. doi: 10.1038/nbt.3811 URL
[60]	Cheng H, Hao M, Ding B, et al. Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system[J]. Plant Biotechnol J, 2021, 19(1): 87-97. doi: 10.1111/pbi.v19.1 URL
[61]	Lin QP, Zong Y, Xue CX, et al. Prime genome editing in rice and wheat[J]. Nat Biotechnol, 2020, 38(5): 582-585. doi: 10.1038/s41587-020-0455-x URL
[62]	Lowder LG, Zhang D, Baltes NJ, et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation[J]. Plant Physiol, 2015, 169(2): 971-985. doi: 10.1104/pp.15.00636 pmid: 26297141
[63]	Moradpour M, Abdulah SNA. CRISPR/dCas9 platforms in plants:strategies and applications beyond genome editing[J]. Plant Biotechnol J, 2020, 18(1): 32-44. doi: 10.1111/pbi.13232 pmid: 31392820
[64]	Duan K, Cheng Y, Ji J, et al. Large chromosomal segment deletions by CRISPR/LbCpf1-mediated multiplex gene editing in soybean[J]. J Integr Plant Biol, 2021, 63(9): 1620-1631. doi: 10.1111/jipb.v63.9 URL
[65]	Huang L, Dong H, Zheng J, et al. Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion[J]. Microbiol Res, 2019, 223/224/225: 44-50.
[66]	Mózsik L, Hoekzema M, de Kok NAW, et al. CRISPR-based transcriptional activation tool for silent genes in filamentous fungi[J]. Sci Rep, 2021, 11(1): 1118. doi: 10.1038/s41598-020-80864-3 URL
[67]	Morio F, Lombardi L, Butler G. The CRISPR toolbox in medical mycology:State of the art and perspectives[J]. PLoS Pathog, 2020, 16(1): e1008201. doi: 10.1371/journal.ppat.1008201 URL

分类 Classifications	物种 Species	Cas9的表达 Expression of Cas9	gRNA的表达 Expression of gRNA	转化材料及递送方式 Materials and delivery methods	编辑模式 Editing methods	靶向基因和筛选标记 Target sites and marker	参考文献 References
担子菌 Basidiomycota	刺芹侧耳 P. eryngii	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	pyrG,cbx^r	2021^[9]
	糙皮侧耳 P. ostreatus	体外合成	体外合成	原生质体,PMT	NHEJ/HDR	pyrG,hyg^r	2021^[10]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	fcy1/pyrG,hyg^r	2021^[11]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	pcc1/clp1,hyg^r	2021^[12]
	双孢蘑菇 A. bisporus	内源gpd启动子表达Cas9	内源U6启动子	菌丝体,ATMT	NHEJ	ppo4,hyg^r	2020^[13]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3/GL17624,bar^r	2020^[14]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	ura3/cyp515018 /cyp505d13,cbx^r	2020^[15]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3,cbx^r	2017^[16]
	裂褶菌 S. commune	外源表达	体外转录	原生质体,PMT	HDR	hom2,nour^r	2019^[17]
	灰盖鬼伞 C. cinerea	内源CcDED1启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	gfp,hyg^r	2017^[18]
	金针菇 F. velutipes	内源gpd启动子表达密码子优化的Cas9	无	菌丝体,ATMT	未编辑	hyg^r	2017^[19]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源gpd启动子	菌丝体,ATMT	未编辑	Fvgpcr1/Fvgpcr2,hyg^r	2019^[20]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源H1启动子	原生质体,PMT	未编辑	hk1/hk2,hyg^r	2018^[21]
子囊菌 Ascomycota	蛹虫草 C. militaris	内源gpd启动子表达密码子优化的Cas9	T7体外转录	分生孢子,ATMT; 原生质体,PMT	NHEJ	ura3,blpR^r	2018^[22]
子囊菌 Ascomycota	竹黄菌 S. bambusicola	内源ef1α启动子表达Cas9	人内源U6启动子	原生质体,PMT	HDR	SbaPKS,hyg^r	2017^[23]

分类 Classifications	物种 Species	Cas9的表达 Expression of Cas9	gRNA的表达 Expression of gRNA	转化材料及递送方式 Materials and delivery methods	编辑模式 Editing methods	靶向基因和筛选标记 Target sites and marker	参考文献 References
担子菌 Basidiomycota	刺芹侧耳 P. eryngii	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	pyrG,cbx^r	2021^[9]
	糙皮侧耳 P. ostreatus	体外合成	体外合成	原生质体,PMT	NHEJ/HDR	pyrG,hyg^r	2021^[10]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	fcy1/pyrG,hyg^r	2021^[11]
	糙皮侧耳 P. ostreatus	来自Coprinopsis cinerea的内源ef3启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	pcc1/clp1,hyg^r	2021^[12]
	双孢蘑菇 A. bisporus	内源gpd启动子表达Cas9	内源U6启动子	菌丝体,ATMT	NHEJ	ppo4,hyg^r	2020^[13]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3/GL17624,bar^r	2020^[14]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ/HDR	ura3/cyp515018 /cyp505d13,cbx^r	2020^[15]
	灵芝 G. lucidum	内源gpd启动子表达密码子优化的Cas9	T7体外转录	原生质体,PMT	NHEJ	ura3,cbx^r	2017^[16]
	裂褶菌 S. commune	外源表达	体外转录	原生质体,PMT	HDR	hom2,nour^r	2019^[17]
	灰盖鬼伞 C. cinerea	内源CcDED1启动子表达密码子优化的Cas9	内源U6启动子	原生质体,PMT	NHEJ	gfp,hyg^r	2017^[18]
	金针菇 F. velutipes	内源gpd启动子表达密码子优化的Cas9	无	菌丝体,ATMT	未编辑	hyg^r	2017^[19]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源gpd启动子	菌丝体,ATMT	未编辑	Fvgpcr1/Fvgpcr2,hyg^r	2019^[20]
	金针菇 F. velutipes	内源gpd启动子表达Cas9	内源H1启动子	原生质体,PMT	未编辑	hk1/hk2,hyg^r	2018^[21]
子囊菌 Ascomycota	蛹虫草 C. militaris	内源gpd启动子表达密码子优化的Cas9	T7体外转录	分生孢子,ATMT; 原生质体,PMT	NHEJ	ura3,blpR^r	2018^[22]
子囊菌 Ascomycota	竹黄菌 S. bambusicola	内源ef1α启动子表达Cas9	人内源U6启动子	原生质体,PMT	HDR	SbaPKS,hyg^r	2017^[23]