供体DNA募集提高塔宾曲霉同源重组效率

doi:10.13560/j.cnki.biotech.bull.1985.2025-1257

摘要/Abstract

摘要：

目的通过mSA-biotin系统将供体DNA募集于基因编辑位点，以提高RNP法介导的CRISPR/Cas9系统在外源基因同源重组中的编辑效率，提高其在丝状真菌无痕基因编辑的效率。方法实验利用单体链霉亲和素（mSA）和生物素的结合能力，首先将mSA与Cas9融合表达，获得重组蛋白Cas9-mSA，并通过5′端生物素标记的通用引物进行PCR扩增，获得生物素标记的供体DNA（Bio-Donor DNA）。将Cas9-mSA和sgRNA在体外组装成有活性的RNP-mSA复合体，并将其与Bio-Donor DNA共同转化进塔宾曲霉F316菌株中，敲除其基因组中的孢子色素相关基因fwnA的同时，以同源重组的方式将绿色荧光蛋白EGFP的表达盒整合进fwnA位点，通过孢子颜色和PCR鉴定快速筛选阳性转化子并统计同源重组效率。结果 Cas9-mSA与Bio-Donor DNA的使用，使供体DNA定向富集于基因编辑位点，使同源重组效率提高了56.53%，促进了RNP法在丝状真菌基因编辑中的应用。结论 Cas9-mSA和Bio-Donor DNA的联合使用有效提高了RNP法在丝状真菌中的同源重组效率。

关键词: Cas9-mSA, Bio-Donor DNA, RNP法, 同源重组效率

Abstract:

Objective The mSA-biotin system was used to recruit donor DNA to target sites, enhancing the editing efficiency of exogenous genes in homology-directed repair (HDR) via RNP-mediated CRISPR/Cas9 and the efficiency in scarless gene editing of filamentous fungi. Method By utilizing the binding ability of streptavidin (mSA) and biotin, mSA was fused with Cas9 for expression, and recombinant protein Cas9-mSA was obtained. Subsequently, donor DNA was amplified by PCR using a 5′-biotinylated primer, yielding biotinylated donor DNA (Bio-Donor DNA). Assembling Cas9-mSA and sgRNA into active RNP-mSA complexes in vitro, and they were co-transformed into Aspergillus tubingensis F316 strain with Bio-Donor DNA. While the spore pigment-related gene fwnA in its genome was knocked out, and the green fluorescent protein EGFP was simultaneously integrated into the fwnA gene site through HDR. Then, the positive transformants were screened through spore color and PCR detection quickly, and HDR efficiency was statistically calculated. Result The use of Cas9-mSA with Bio-donor DNA makes the donor DNA directly enriched toward gene editing sites, and thus HDR efficiency increased by 56.53%, which promoted the application of RNP method in gene editing of filamentous fungi. Conclusion The combined use of Cas9-mSA and Bio-Donor DNA effectively enhances the HDR efficiency of the RNP method in filamentous fungi.

Key words: Cas9-mSA, Bio-Donor DNA, RNP method, homology-directed repair (HDR) efficiency

卢春秋, 梁丽存, 王苑, 黄火清, 罗会颖, 杨浩萌. 供体DNA募集提高塔宾曲霉同源重组效率[J]. 生物技术通报, 2026, 42(8): 90-96.

LU Chun-qiu, LIANG Li-cun, WANG Yuan, HUANG Huo-qing, LUO Hui-ying, YANG Hao-meng. Donor DNA Recruitment Improving the Homology-directed Repair Efficiency in Aspergillus tubingensis[J]. Biotechnology Bulletin, 2026, 42(8): 90-96.

图/表 6

表1 引物名称及序列信息

Table 1 Primer names and sequence information

引物名称 Primer name	序列 Sequence （5′-3′）
fwn-up-500-F	TCAACCAAGCTCCGTGTCCCTTACGC
fwn-up-R	GTTCTTGAGATCCCACTTGTAGGCTGGG
fwn-dw-F	TACTGGATTCCCTATACCAACAACTTCTGC
fwn-dw-500-R	CGCAATCAAATAATCGGACAGTGC
up-fwn-1k-F	CTCTACCGCAGTTGTTTCCAGTTTCG
HPH-F	AAGTGGGATCTCAAGAACTCGACAGAAGATGATATTGAAGGAGCAC
HPH-R	CCTCGCCCTTGCTCACCATGTATCTGGAAGAGGTAAACCCGAAACG
EGFP-F	GCATGCGGAGAGACGGACGG
EGFP-R	TGCATTGCAGATGAGCTGTATCTGG
BIO-F	GACTCATGTTGGTATTGTGAAATAGACGCAGATCGGG
BIO-R	CTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC
BIO-fwn-2.6-F	GGTATTGTGAAATAGACGCAGATCGGGTCAACCAAGCTCCGTGTCCCTTACGC
BIO-fwn-2.6-R	GAGCTGCATGTGTCAGAGGTTTTCACCCGCAATCAAATAATCGGACAGTGC
EGFP-JC-F	CGGGATCACTCTCGGCATGGAC
500-down-JC-R	GTGCTGTTCACTGTCAAGAATAACCTCGCG
HYG-JC-R	GGCGGGAGATGCAATAGGTCAG
2k-up-JC-F	TGAAGCCGGCCTTCATTATCGG

图1 重组Cas9-mSA蛋白纯化组分的SDS-PAGE分析（A）和RNP复合体体外活性验证（B）A： M：Marker蛋白；1：诱导后细胞破碎液上清；2：10 mmol/L咪唑洗涤液；3：20 mmol/L咪唑洗涤液；4：40 mmol/L咪唑洗涤液；5：100 mmol/L咪唑洗脱液；6：500 mmol/L咪唑洗脱液。B：1：对照DNA；2-4：3种RNP复合体体外活性验证情况

Fig. 1 SDS-PAGE analysis of purified recombinant Cas9-mSA protein (A) and in vitro activity assay of RNP complexes (B)A: M: Marker protein; 1: supernatant of cell lysate after induction; 2: wash with 10 mmol/L imidazole; 3: wash with 20 mmol/L imidazole; 4: wash with 40 mmol/L imidazole; 5: elution with 100 mmol/L imidazole; 6: elution with 500 mmol/L imidazole. B: 1: Control DNA; 2-4: three kinds of RNP complex in vitro activity verification

图2 实验组和对照组的转化子颜色变化

Fig. 2 Color change of transformants in the experimental group and control group

图3 同源重组阳性转化子的PCR验证及测序结果A：对照Cas9组同源重组转化子的PCR检测结果；B：Cas9-mSA组同源重组转化子的PCR检测结果；C：测序结果，蓝色碱基序列为gRNA的特异识别序列，1#-5#为基因编辑阳性转化子测序序列

Fig. 3 PCR verification and sequencing results of homologous recombination positive transformantsA: Results of PCR detection of HDR transformants of control Cas9 group. B: The result of PCR detection of HDR efficiency of Cas9-mSA transformants. C: Sequencing result. The blue base sequence is the specific recognition sequence of gRNA, and 1#-5# are the positive gene edited sequences

表2 实验组及对照组效率统计

Table 2 Efficiency statistics of experimental group and control group

组别

Treatment

被编辑菌株/总数

Edited strain/Total

基因敲除效率

Gene-knockout efficiency （%）

外源基因同源重组效率

HDR efficiency of exogenous genes （%）

图4 菌株荧光显微镜下的明暗视野

Fig. 4 Strains in the bright and excitation vision of fluorescence microscope

参考文献 31

[1]	Deng HX, Gao RJ, Liao XR, et al. CRISPR system in filamentous fungi: Current achievements and future directions [J]. Gene, 2017, 627: 212-221.
[2]	Jin FJ, Wang BT, Wang ZD, et al. CRISPR/Cas9-based genome editing and its application in Aspergillus species [J]. JoF, 2022, 8(5): 467.
[3]	Scully R, Panday A, Elango R, et al. DNA double-strand break repair-pathway choice in somatic mammalian cells [J]. Nat Rev Mol Cell Biol, 2019, 20(11): 698-714.
[4]	Fuller KK, Chen S, Loros JJ, et al. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus [J]. Eukaryot Cell, 2015, 14(11): 1073-1080.
[5]	Meyer V, Arentshorst M, El-Ghezal A, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant [J]. J Biotechnol, 2007, 128(4): 770-775.
[6]	Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system [J]. Nat Protoc, 2013, 8(11): 2281-2308.
[7]	梁丽存, 刘文龙, 刘晓青, 等. 化学试剂对塔宾曲霉同源重组效率的影响 [J]. 生物技术进展, 2024, 14(4): 586-593.
	Liang LC, Liu WL, Liu XQ, et al. The effect of chemical reagents on the efficiency of gene editing in Aspergillus tubingensis [J]. Curr Biotechnol, 2024, 14(4): 586-593.
[8]	Gandía M, Xu SM, Font C, et al. Disruption of Ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum [J]. Fungal Biol, 2016, 120(3): 317-323.
[9]	Fang Z, Zhang Y, Cai MH, et al. Improved gene targeting frequency in marine-derived filamentous fungus Aspergillus glaucus by disrupting ligD [J]. J Appl Genetics, 2012, 53(3): 355-362.
[10]	杨浩萌, 梁丽存, 宋祖洹, 等. 提高丝状真菌精准基因编辑效率的策略 [J]. 微生物学通报, 2024, 51(5): 1425-1440.
	Yang HM, Liang LC, Song ZH, et al. Strategies for improving the efficiency of precise gene editing in filamentous fungi [J]. Microbiol China, 2024, 51(5): 1425-1440.
[11]	Laitinen OH, Hytönen VP, Nordlund HR, et al. Genetically engineered avidins and streptavidins [J]. Cell Mol Life Sci, 2006, 63(24): 2992-3017.
[12]	Kuntz ID, Chen K, Sharp KA, et al. The maximal affinity of ligands [J]. Proc Natl Acad Sci U S A, 1999, 96(18): 9997-10002.
[13]	Wilchek M, Bayer EA, Livnah O. Essentials of biorecognition: The (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction [J]. Immunol Lett, 2006, 103(1): 27-32.
[14]	Pähler A, Hendrickson WA, Kolks MA, et al. Characterization and crystallization of core streptavidin [J]. J Biol Chem, 1987, 262(29): 13933-13937.
[15]	Le Q, Nguyen V, Park S. Recent advances in the engineering and application of streptavidin-like molecules [J]. Appl Microbiol Biotechnol, 2019, 103(18): 7355-7365.
[16]	冯爽, 王春伟, 苏小虎. 动物基因组编辑中提升CRISPR/Cas9介导的同源重组效率研究进展 [J]. 中国生物工程杂志, 2022, 42(9): 83-92.
	Feng S, Wang CW, Su XH. Research advancement of CRISPR/Cas9 directed homologous recombination efficiency improvements in mammal genome editing [J]. China Biotechnol, 2022, 42(9): 83-92.
[17]	Roche PJR, Gytz H, Hussain F, et al. Double-stranded biotinylated donor enhances homology-directed repair in combination with Cas9 monoavidin in mammalian cells [J]. CRISPR J, 2018, 1(6): 414-430.
[18]	Carlson-Stevermer J, Abdeen AA, Kohlenberg L, et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing [J]. Nat Commun, 2017, 8: 1711.
[19]	Shechner DM, Hacisuleyman E, Younger ST, et al. Multiplexable, locus-specific targeting of long RNAs with CRISPR-display [J]. Nat Methods, 2015, 12(7): 664-670.
[20]	Leppek K, Stoecklin G. An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins [J]. Nucleic Acids Res, 2014, 42(2): e13.
[21]	Kheirandish MH, Rahmani B, Zarei Jaliani H, et al. Efficient site-specific integration in CHO-K1 cells using CRISPR/Cas9-modified donors [J]. Mol Biol Rep, 2023, 50(7): 5889-5899.
[22]	梁丽存, 于会民, 管晓晨, 等. 塔宾曲霉的鉴定及基因编辑方法对其同源重组效率的影响 [J]. 中国农业科技导报, 2025, 27(6): 83-92.
	Liang LC, Yu HM, Guan XC, et al. Identification of Aspergillus tubingensis and the effect of gene editing methods on its homologous recombination efficiency [J]. J Agric Sci Technol, 2025, 27(6): 83-92.
[23]	Huang DM, Song YY, Liu YL, et al. A new strain of Aspergillus tubingensis for high-activity pectinase production [J]. Braz J Microbiol, 2019, 50(1): 53-65.
[24]	Okado N, Sugi M, Kasamoto S, et al. Safety evaluation of arabinase (arabinan endo-1, 5-α-L-arabinanase) from Aspergillus tubingensis [J]. Food Sci Nutr, 2020, 8(1): 456-478.
[25]	Krisnawati R, Cahyanto MN, Sardjono S, et al. RNA-seq data of Aspergillus tubingensis NBRC 31125 in carbon catabolite repressor related to xylanase production [J]. Data Brief, 2022, 45: 108700.
[26]	Wu YM, Yang XQ, Zhao TD, et al. Antifeedant and antifungal activities of metabolites isolated from the coculture of endophytic fungus Aspergillus tubingensis S1120 with red ginseng [J]. Chem Biodivers, 2022, 19: e202100608.
[27]	郭雷, 王聪, 郭家才, 等. 海洋来源塔宾曲霉LWG-42菌株的鉴定及其抗氧化活性化合物的分离 [J]. 微生物学杂志, 2017, 37(6): 30-35.
	Guo L, Wang C, Guo JC, et al. Identification of marine-derived Aspergillus tubingensis LWG-42 and isolation of its antioxidant active compounds [J]. J Microbiol, 2017, 37(6): 30-35.
[28]	孔华忠, 齐祖同. 曲霉属和散囊菌属的新记录 [J]. 真菌学报, 1995, 14(1): 75-78.
	Kong HZ, Qi ZT. New records of Aspergillus and Eurotium in Chian [J]. Mycosystema, 1995, 14(1): 75-78.
[29]	高伟欣, 黄火清, 赵晶, 等. 应用于基因编辑的核糖核蛋白复合体的构建与活性验证 [J]. 生物技术通报, 2022, 38(8): 60-68.
	Gao WX, Huang HQ, Zhao J, et al. Construction and activity verification of ribonucleoprotein complex for gene editing [J]. Biotechnol Bull, 2022, 38(8): 60-68.
[30]	Ma M, Zhuang FF, Hu XB, et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system [J]. Cell Res, 2017, 27(4): 578-581.
[31]	Gu B, Posfai E, Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos [J]. Nat Biotechnol, 2018, 36(7): 632-637.