Improving the Efficiency of Gene Editing by Optimizing sgRNA in Aspergillus tubingensis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0784

Abstract

Abstract:

Objective In the CRISPR/Cas9 gene editing system, sgRNA is one of the important gene editing elements. It binds to the Cas9 protein and complements the genomic DNA through the spacer sequence to guide the Cas9 protein to accurately cut the genome. In order to improve the gene editing efficiency of Aspergillus tubingensis, sgRNA optimization is a feasible strategy. Method The promoter and hairpin structure of sgRNA were optimized, and the gene editing efficiency was verified in A. tubingensis. Result The gene editing efficiency of sgRNA with “lock” structure was 9.37% higher than that of the control group when gene editing was performed by RNP method. When sgRNA was expressed in vivo, the gene editing efficiency of tRNA^Gly15 and tRNA^Gly17 promoters was 14%-16% higher than that of 5S rRNA promoter, respectively. Using the tRNA^Gly15 promoter to express sgRNA with a “lock” structure increased the white spore rate and increased the gene editing efficiency of A. tubingensis to 96%. Conclusion The “lock” structure and two promotors of tRNA^Gly15 and tRNA^Gly17 may improve the gene editing efficiency of A. tubingensis.

Key words: Aspergillus tubingensis, gene editing efficiency, promoter, “lock” structure

LIANG Li-cun, WANG Ke-fen, SONG Zu-huan, LIU Meng-ting, LI Jia-yu, LUO Hui-ying, YAO Bin, YANG Hao-meng. Improving the Efficiency of Gene Editing by Optimizing sgRNA in Aspergillus tubingensis[J]. Biotechnology Bulletin, 2025, 41(3): 62-70.

Figures/Tables 7

Fig. 1 Schematic diagram of gene editing element structure and editing processA: The left side from top to bottom indicates the composition of cas9 expression cassette, sgRNA expression cassette of different promoters and donor DNA, respectively. The Cas9 expression cassette is composed of Pterf promoter, coding sequence, nuclear localization signal peptide NLS and Tterf terminator. The promoters of sgRNA are T7 promoter, 5SrRNA promoter (including 134 bp and 395 bp), tRNAGly15 promoter and tRNAGly17 promoter, respectively. The tracrRNA skeleton of normal sgRNA is in the blue dotted box above the right side, and the sgRNA tracrRNA skeleton in the dotted box below carries a hairpin structure represented by a “lock” graph. Donor DNA consists of 500 bp upstream and downstream homologous arms and intermediate hygromycin resistance genes. B: A brief schematic diagram of the CRISPR/Cas9 system, which shows the editing originals required for the expression cassette method and the RNP method. When the expression cassette method is used for transformation, the Cas9 expression cassette and sgRNA expression cassette need to be added to the prepared protoplasts together with Donor DNA. When the RNP method was used for transformation, Cas9 protein and sgRNA were incubated in vitro, and the two were added to the prepared protoplasts together with Donor DNA in the form of RNP complex for transformation. The target gene fwnA was cut, and the genome sequences on both sides of the cutting site were homologously recombined with the homologous arm of Donor DNA, thus the hygromycin gene was inserted into the fwnA gene, and the gene was inactivated, resulting in the spore color from black to white, and the gene detection primers were labeled on the schematic diagram of Donor DNA

Table 1 Primers used in this study

引物名称Primer name	序列Sequence
fwn-up-500-F	5'-CAACCAAGCTCAAGGTTCCTTACGCG-3'
fwn-up-R	5'-GTTCTTGAGATCCCACTTGTAGGCTGGG-3'
fwn-dw-F	5'-CTATACCAACAACTTCTGCCTGAGCAAGG-3'
fwn-dw-500-R	5'-TCCGCAGCAGCGCAGTCAAAG-3'
hph-fu-F	5'-CAGGCTACAAGTGGGATCTCAAGAACGACAGAAGATGATATTGAAGGAGCACTTTTTG-3'
hph-fu-R	5'-AGTTGTTGGTATAGGGAATCCAGTAGTATCTGGAAGAGGTAAACCCGAAACG-3'
5SRNA-F1	5'-ACGAAGAGGATGGTTGAACACGGAT-3'
5SRNA-F2	5'-CACATACGACCACAGGGTGTG-3'
P5SRNA-R	5'-ACATACAACAGAAGGGATTCGCTGGTG-3'
5SRNA-overlap-F	5'-CACCAGCGAATCCCTTCTGTTGTATGTAGTGGGATCTCAAGAACTACGTTTTAGAGC-3'
sgRNA-R	5'-AAAAGCACCGACTCGGTGCC-3'
CAS9-F	5'-CGAGACAGCAGAATCACCGCCCAAGTTAAG-3'
CAS9-R	5'-ATTACACTTGTATTGGGATGAATTTTGTATGCAC-3'
tRNA^Gly15-F	5'-CTCCGTAGATAGAGATAGGAGTGGATAGGGA-3'
tRNA^Gly15-R	5'-TGCATCATCCGTGAATCGAACACGG-3'
tRNA^Gly17-F	5'-ACAATTCACAATCCGCAAGACGTTAACG-3'
tRNA^Gly17-R	5'-TGCATCATCCGTGAATCGAACACGG-3'
tRNA^Gly15/17-overlap-F	5'-CCGTGTTCGATTCACGGATGATGCAAGTGGGATCTCAAGAACTACGTTTTAG-3'

Table 2 Sequence information of sgRNA in vitro transcription template

名称Name	序列Sequence（5'-3'）
PT7-sgRNA-fwn	TAATACGACTCACTATAGGAGTGGGATCTCAAGAACTACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
PT7-lock-sgRNA-fwn	TAATACGACTCACTATAGGAGTGGGATCTCAAGAACTACGTTTTAGAGCTATGCTAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGACTTCGGTCCAAGTGGCACCGAGTCGGTGCTTTT

Fig. 2 Amplification of Cas9 expression cassette, sgRNA expression cassette and Donor DNA1: sgRNA with 134 bp 5S rRNA as promoter; 2: sgRNA with 395 bp 5S rRNA as promoter; 3: a sgRNA with tRNAGly17 as the promoter; 4: a sgRNA with tRNAGly15 as the promoter; 5: sgRNA with tRNAGly15 as promoter and fused with “lock” structure; 6: Donor DNA; 7:Cas9 expression cassette

Fig. 3 Growth of transformants and the gene editing efficiency of different sgRNA promoters during transformation by expression cassette methodA: The growths of transformants in the two experimental groups of 5S rRNA 134 bp and tRNAGly15 (the colors of black spores, white spores, and miscellaneous spores are shown in the figure). B: The gene editing efficiency of sgRNA with different lengths of 5S promoter, tRNAGly15 promoter and tRNAGly17 promoter

Fig. 4 Gene editing efficiency of sgRNA with “lock” structure in RNP methodThe histogram in Fig.4 shows the gene editing efficiency of sgRNA containing ordinary sgRNA structure and sgRNA fused with “lock” structure.Three biological replicates were performed in each experimental group, and the obtained data were analyzed by spss software. The error line indicates the size of the standard error.The significance level between different letters is 0.05 ( the same below )

Fig. 5 Gene editing efficiency of tRNAGly15 promoter superimposed with “lock” structure in expression cassette methodThe gene editing efficiency of sgRNA with tRNAGly15 as the promoter and sgRNA with tRNAGly15 as the promoter and fused with “lock” structure

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