Donor DNA Recruitment Improving the Homology-directed Repair Efficiency in Aspergillus tubingensis

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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

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 （%）

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

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