Cytosine base editing plasmid, base editing system and application

Abstract

The invention belongs to the technical field of microbial genetic engineering and gene editing. The invention provides a Cas9 enzyme mediated cytosine base editing plasmid, and compared with a starting plasmid, the Cas9 enzyme mediated cytosine base editing plasmid has the following inserted sequences: a terminator Tcyc1; the base sequence of the terminator Tcyc1 is as shown in SEQ ID NO. 5; a promoter PgpdA; the base sequence of the promoter PgpdA is as shown in SEQ ID NO. 1; the preparation method comprises the following steps: preparing a mini-SDD7 deaminase; the amino acid sequence of the mini-SDD7 deaminase is as shown in SEQ ID NO. 2; an nCas9 (D10A) coding sequence is obtained; the amino acid sequence of the nCas9 (D10A) coding sequence is as shown in SEQ ID NO. 3; a UGI coding sequence; the amino acid sequence of the UGI coding sequence is as shown in SEQ ID NO. 4; the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg, and the starting plasmid is a plasmid pUC19-AMA1-(P) Hyg. The base editing system provided by the invention can be used for gene editing in trichoderma koningii.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and gene editing technology. Technical Background

[0002] Trichoderma fungi are a group of multifunctional filamentous fungi. Most Trichoderma species are used in agricultural production as biocontrol agents due to their biocontrol properties. Furthermore, Trichoderma can produce and secrete various cellulase and hemicellulase systems, which are widely used in research on lignocellulose degradation. Among them, *Trichoderma koningiopsis* has attracted much attention in the field of industrial enzyme production due to its excellent cellulase and hemicellulase production capabilities. However, due to the multicellular structure of filamentous fungi, *Trichoderma* fungi have a more complex genetic background compared to single-celled organisms, making genetic manipulation difficult. Therefore, targeted genetic modification to rationally improve strain performance, enhance endogenous gene expression, and improve enzyme production characteristics presents significant challenges. Developing an efficient gene editing system is of great importance for both basic and applied research in *Trichoderma* fungi. In recent years, the CRISPR / Cas9 system based on homologous recombination (HDR) has been successfully applied in *T. reesei*. [1] This technology enables highly efficient gene knockout and insertion. While using Cas9 to generate DNA double-strand breaks (DSBs) to improve editing efficiency, random mutations caused by non-homologous end joining (NHEJ) are unavoidable. Single-base editing technology was introduced in 2016. [2] This technology leverages the precise DNA localization capabilities of the CRISPR / Cas9 system and the highly efficient catalytic ability of base deaminases for precise editing. Compared to HDR-based gene editing technologies, base editing does not rely on the inefficient DNA repair process of HDR, nor does it utilize Cas9 to cause DNA double-strand breaks, thus avoiding random mutations caused by NHEJ. Therefore, it is generally more precise and efficient. Establishing an efficient base editing system in filamentous fungi such as *Trichoderma tumefaciens* and utilizing this system for efficient single / multi-gene editing is of great significance. Summary of the Invention

[0003] In existing technologies, precise editing of filamentous fungal genomes faces problems such as low efficiency, high off-target effects, and plasmid integration, affecting the genetic stability of strains and subsequent industrial applications. Therefore, this invention provides a Cas9 enzyme-mediated cytosine base editing plasmid, which, compared to the starting plasmid, inserts the following sequences: a terminator Tcyc1; the base sequence of the terminator Tcyc1 is shown in SEQ ID NO. 5; a promoter PgpdA; the base sequence of the promoter PgpdA is shown in SEQ ID NO. 1; a mini-SDD7 deaminase; the amino acid sequence of the mini-SDD7 deaminase is shown in SEQ ID NO. 2; an nCas9 (D10A) coding sequence; the amino acid sequence of the nCas9 (D10A) coding sequence is shown in SEQ ID NO. 3; a UGI coding sequence; the amino acid sequence of the UGI coding sequence is shown in SEQ ID NO. 4; and the starting plasmid is plasmid pUC19-AMA1-Hyg.

[0004] The present invention also provides a base editing system, comprising the aforementioned cytosine base editing plasmid and sgRNA expression cassette; the sgRNA expression cassette comprises a glycine promoter tRNA, an sgRNA sequence of a target sequence designed for the edited target gene, and a 6-T terminator; the base sequence of the glycine promoter tRNA is shown in SEQ ID NO. 6.

[0005] In a specific embodiment of the present invention, the target gene is ura5; the base sequence of the sgRNA sequence is shown in SEQ ID NO.7.

[0006] In a specific embodiment of the present invention, the target gene is yak1; the base sequence of the sgRNA sequence is shown in SEQ ID NO.8.

[0007] In a specific embodiment of the present invention, the target genes are ace1 and yak1; the base sequence of the sgRNA sequence of ace1 is shown in SEQ ID NO.9; the base sequence of the sgRNA sequence of yak1 is shown in SEQ ID NO.8; and the sgRNA expression cassette is two sgRNA expression cassettes tandem.

[0008] In a specific embodiment of the present invention, the target genes are pk1, ace1, and yak1; the base sequence of the sgRNA sequence of pk1 is shown in SEQ ID NO.10; the base sequence of the sgRNA sequence of ace1 is shown in SEQ ID NO.9; the base sequence of the sgRNA sequence of yak1 is shown in SEQ ID NO.8; and the sgRNA expression cassette is three sgRNA expression cassettes tandem.

[0009] The base editing system provided by this invention enables gene editing in Trichoderma synergii.

[0010] The beneficial effects achieved by this invention are as follows:

[0011] The base editing system for Trichoderma tumefaciens strains provided by this invention can construct a highly efficient Trichoderma tumefaciens gene editing platform, achieving editing efficiencies of 15-90% for single genes and 10-13% for multiple genes in the obtained transformants. The mini-SDD7 editing window is relatively compact, allowing for fine-tuning of target codons by adjusting the relative positions of the target site PAM, facilitating codon optimization and key amino acid function verification. In terms of improving the production performance of industrial strains, precise fine-tuning of multiple sites in regulatory genes makes it easier to maintain strain growth robustness than "major surgical" knockout, which is beneficial for scale-up culture and enzyme production curve optimization. In summary, the self-replicating mini-SDD7-CBE base editing system provided by this invention significantly outperforms existing traditional fungal genome modification strategies in multiple dimensions, including removability, accuracy, scalability, efficiency, safety, and industrial commercialization potential. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described in detail below.

[0013] Figure 1 pUC19-AMA1-Hyg-Tcyc1 plasmid map and its enzyme digestion verification.

[0014] (A) Map of plasmid pUC19-AMA1-Hyg-Tcyc1; (B) Enzyme digestion verification of plasmid pUC19-AMA1-Hyg-Tcyc1. M: DNA marker. 1: Plasmid pUC19-AMA1-Hyg-Tcyc1 digested with HindIII yielded three fragments, consistent with the theoretical size.

[0015] Figure 2 The plasmid map of pUC19-AMA1-Hyg-PgpdA-miniSDD7-Tcyc1 and its enzyme digestion verification were obtained.

[0016] (A) Map of pUC19-AMA1-Hyg-PgpdA-miniSDD7-Tcyc1; (B) Enzyme digestion verification of pUC19-AMA1-Hyg-PgpdA-miniSDD7-Tcyc1. M: DNA marker. 1: The plasmid pUC19-AMA1-Hyg-PgpdA-miniSDD7-Tcyc1, digested with HindIII, yielded four fragments, consistent with the theoretical size.

[0017] Figure 3 The plasmid map of pUC19-AMA1-Hyg-PgpdA-miniSDD7-nCas9-UGI-Tcyc1 and its enzyme digestion verification were obtained.

[0018] (A) Map of pUC19-AMA1-Hyg-PgpdA-miniSDD7-nCas9-UGI-Tcyc1; (B) Enzyme digestion verification of pUC19-AMA1-Hyg-PgpdA-miniSDD7-nCas9-UGI-Tcyc1. M: DNA marker. 1: Five fragments were obtained from the plasmid pUC19-AMA1-Hyg-PgpdA-miniSDD7-nCas9-UGI-Tcyc1 digested with HindIII, which is consistent with the theoretical size.

[0019] Figure 4 This is a schematic diagram of the structure of plasmid CBE-sgRNA.

[0020] Figure 5 This document describes the editing status of the ura5 gene target site in the transformant.

[0021] The PAM sequence is highlighted in green. Expected editing sites are indicated by nucleotide numbers relative to the PAM sequence and shaded in gray. The corresponding translated amino acid sequence is listed below each nucleotide sequence. Mutant bases and amino acids are highlighted in red.

[0022] Figure 6 Status of yak1 gene target site editing.

[0023] Figure 7 A schematic diagram of sgRNA construction for simultaneous editing of the ace1 and yak1 genes.

[0024] Figure 8 Editing status of the ace1 and yak1 gene target sites in the transformants.

[0025] Figure 9 Editing status of gene target sites in transformants pk1, ace1, and yak1.

[0026] Figure 10 PCR validation results of plasmid elimination targeting yak1 gene editing transformant. Detailed Implementation

[0027] The following examples further illustrate the construction, application, and multi-gene simultaneous editing method of the *Trichoderma tumefaciens* base editing system based on mini-SDD7-CBE described in this invention. Those skilled in the art can make equivalent substitutions and appropriate adjustments to conditions, proportions, and sequences without departing from the spirit of this invention, and all such substitutions and adjustments should be considered to fall within the protection scope of this invention.

[0028] General Materials and Methods

[0029] Table 1. Strains used in this invention

[0030]

[0031] Table 2. Plasmids used in this invention

[0032]

[0033] Table 3 Primers used in this invention

[0034]

[0035] PDA medium (g / L): 200 g potato, 20 g glucose. Cut 200 g peeled potatoes into small pieces, boil in boiling water for 30 min, filter through eight layers of gauze, dissolve glucose in the filtrate, and then add distilled water to a final volume of 1 L. PDA solid medium requires the addition of 1.5% agar powder; without agar powder, it becomes PDB medium. The pH should be natural. Autoclave at 115℃ for 30 min.

[0036] Protoplast transformation supernatant medium (g / L): 39 g potato broth, 218.6 g sorbitol, 0.5% agar powder, pH natural. Autoclaved at 115℃ for 30 min.

[0037] Protoplast transformation substrate medium (g / L): 39 g potato broth, 218.6 g sorbitol, 1.5% agar powder, pH natural. Autoclaved at 115°C for 30 min.

[0038] CTAB buffer: CTAB 1% (w / v); EDTA (pH 8.0) 0.02 M; Tris-HCl (pH 8.0) 0.1 M; NaCl 1.4 M; β-mercaptoethanol 1% (v / v).

[0039] Osmotic Medium: MgSO4·7H2O 1.2 M; phosphate buffer (pH 6.5) 0.01 M, adjusted to pH 5.8 using 1 M Na2HPO4.

[0040] Trapping buffer: Sorbitol 0.6 M; Tris-HCl (pH 7.5) 0.1 M.

[0041] STC buffer: Sorbitol 1.2 M; CaCl2 0.01 M; Tris-HCl (pH 7.5) 0.01 M.

[0042] PEG buffer: PEG4000 60% (w / v); CaCl2 0.05 M; Tris-HCl (pH 7.5) 0.05 M.

[0043] Antibiotic use: Hygromycin B screening concentration 200 μg / mL; 5′-FOA reverse screening concentration 0.8–1.2 g / L, supplemented with uracil or uridine (50–100 mg / L).

[0044] Enzyme preparations: Restriction endonucleases (purchased from Baori Biotechnology Co., Ltd.), Gibson assembly reagents (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.), and high-fidelity PCR polymerases (purchased from Nanjing Novizan Biotechnology Co., Ltd.) were all commercially available.

[0045] Sequencing validation: Sanger sequencing (Qingke Biotechnology), bidirectional primers covering the editing window ±120 bp region.

[0046] Example 1

[0047] Constructing a fusion-based mini-SDD7-CBE main expression framework

[0048] We used the laboratory-preserved plasmid pUC19-AMA1-(P)Hyg as the starting plasmid. [4] An expression plasmid containing the encoding gene of a fusion protein derived from bacterial cytosine deaminase (mini-SDD7), nCas9 (D10A), and uracil DNA glycosylation inhibitor (UGI) was constructed.

[0049] First, the terminator Tcyc1 sequence was added to the starting plasmid pUC19-AMA1-(P)Hyg to prepare the first intermediate plasmid pUC19-AMA1-Hyg-Tcyc1.

[0050] The linearized plasmid was obtained by digesting the starting plasmid pUC19-AMA1-(P)Hyg with KpnI enzyme.

[0051] To build a nest Aspergillus genome is Using a template, primers Tcyc1-F / Tcyc1-R were designed, and a DNA fragment containing the terminator Tcyc1 was obtained by PCR amplification. The base sequence of the terminator Tcyc1 is shown in SEQ ID NO.5.

[0052] The DNA fragment containing the terminator Tcyc1 was ligated to the linearized starter plasmid pUC19-AMA1-Hyg using the Gibson assembly method, resulting in the first intermediate plasmid pUC19-AMA1-Hyg-Tcyc1 with the added terminator Tcyc1 sequence. Digestion of this plasmid with HindIII yielded three fragments, consistent with the theoretical size. Figure 1 Further sequencing confirmed that the plasmid was constructed correctly.

[0053] Subsequently, based on the first intermediate plasmid pUC19-AMA1-Hyg-Tcyc1, the promoter PgpdA and the coding sequences of mini-SDD7, which drive the expression of effector protein genes, were inserted. A nuclear localization signal (NLS) was also designed and added to the carboxyl terminus of the mini-SDD7 deaminase to prepare the second intermediate plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-Tcyc1. The base sequence of the promoter PgpdA is shown in SEQ ID NO.1. The amino acid sequence of the mini-SDD7 deaminase is shown in SEQ ID NO.2.

[0054] The linearized plasmid was obtained by digesting the first intermediate plasmid pUC19-AMA1-Hyg-Tcyc1 with KpnI. The plasmid pUC19-AMA1-Hyg-Cas9 was then used as the linearized plasmid. [4] Using a template, primers PgpdA2-F / PgpdA2-R were designed to amplify DNA fragments containing the promoter PgpdA sequence via PCR.

[0055] The gene sequence encoding mini-SDD7 deaminase containing the nuclear localization signal NLS at the carboxyl terminus was synthesized by Nanjing GenScript Biotech Co., Ltd. after optimization according to the codon preferences of Trichoderma fungi. The company then inserted this genome into the plasmid pUC57-SDD7. Using this plasmid as a template, primers sdd7-F / sdd7-R were designed, and the DNA fragment encoding the mini-SDD7 sequence containing the nuclear localization signal NLS at the carboxyl terminus was obtained by PCR amplification.

[0056] The two fragments were ligated to linearized pUC19-AMA1-Hyg-Tcyc1 using the Gibson assembly method to obtain the second intermediate plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-Tcyc1. Digestion of this plasmid with HindIII yielded four fragments, consistent with the theoretical size. Figure 2 Further sequencing confirmed that the plasmid was constructed correctly.

[0057] Third, based on the second intermediate plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-Tcyc1, the coding sequence for the effector protein element nCas9 (D10A) was inserted. [5] Uracil DNA glycosylation inhibitor UGI coding sequence [3] The cytosine base editing plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-nCas9-UGI-Tcyc1 was prepared using the nuclear localization signal (NLS) coding sequence. The amino acid sequence of the effector protein element nCas9 (D10A) is shown in SEQ ID NO.3. The amino acid sequence of the uracil DNA glycosyltransferase inhibitor UGI is shown in SEQ ID NO.4.

[0058] Linearized plasmids were obtained by digesting the second intermediate plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-Tcyc1 with PacI. The nCas9 (D10A) coding gene sequence, containing a nuclear localization signal (NLS) at both the carboxyl and amino ends, was synthesized by Nanjing GenScript Biotech Co., Ltd. after optimization according to the codon preferences of Trichoderma fungi. The company then inserted this genome into plasmid pUC57-nCas9. Primers Cas-F / Cas-R were designed using pUC57-nCas9 as a template, and DNA fragments encoding the nCas9 (D10A) coding sequence with nuclear localization sequences at both ends were obtained by PCR amplification. The UGI-coding gene sequence with a nuclear localization signal (NLS) at the amino terminus was synthesized by Nanjing GenScript Biotech Co., Ltd. after optimization according to the codon preferences of Trichoderma fungi. The company then inserted this genome into the plasmid pUC57-nCas9. Using pUC57-nCas9 as a template, primers ugi-F / ugi-R were designed, and DNA fragments containing the UGI coding sequence were obtained by PCR amplification. This fragment was then ligated to the linearized pUC19-AMA1-Hyg-PgpdA-miniSDD7-Tcyc1 using the Gibson assembly method, resulting in the cytosine base editing plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-nCas9-UGI-Tcyc1 containing the base editor mini-SDD7-CBE. Digestion of this plasmid with HindIII yielded 5 fragments, consistent with the theoretical size. Figure 3 Further sequencing confirmed that the plasmid was constructed correctly.

[0059] Example 2

[0060] Construction of tRNA(Gly)-sgRNA expression framework

[0061] Based on the constructed plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-nCas9-UGI-Tcyc1 containing the cytosine base editor, an sgRNA expression cassette was designed and constructed. In CRISPR / Cas9-based gene editing systems, sgRNA is an indispensable core component, and the selection of the sgRNA promoter is crucial for its efficient expression. A glycine-containing tRNA promoter from *Trichoderma tegmentans* was selected, primers were designed, and PCR amplification was performed. Using *Trichoderma tegmentans* genomic DNA as a template, primers tRNA-F / tRNA-R were designed to amplify a DNA fragment containing the glycine promoter tRNA (Gly). The base sequence of the glycine promoter tRNA is shown in SEQ ID NO. 6.

[0062] The constructed base editing system's sgRNA expression cassette consists of three parts: a promoter driving sgRNA expression (glycine-based promoter tRNA), a dimer (sgRNA) composed of a 20 bp crRNA targeting the target gene and a trans-activating RNA (tracrRNA) that can be recognized by the nCas9 protein, and a six-T terminator to terminate sgRNA expression. The 20 bp crRNA targeting the target gene was designed using the online website CRISPOR (https: / / crispor.gi.ucsc.edu / crispor.py), selecting the highest-ranking crRNA.

[0063] Generally, in vivo transcription of sgRNA is a more stable method and is more suitable for gene editing in filamentous fungi. To improve the efficiency of gene editing in *Trichoderma tumefaciens*, the constructed cytosine base editor plasmid and sgRNA expression cassette were assembled together and then transformed into *Trichoderma tumefaciens*.

[0064] First, the cytosine base editing plasmid pUC19-AMA1-Hyg-PgpdA-mini-SDD7-nCas9-UGI-Tcyc1 was linearized using KpnI. Then, the sgRNA expression cassette was amplified by fusion PCR, and finally, the final base editing system was constructed using Gibson assembly. Figure 4 ).

[0065] Example 3

[0066] Targeted editing of ura5

[0067] ura5 encodes orotate nucleoside phosphotransferase, a key enzyme in the synthesis of uracil nucleotides. Deletion of the ura5 gene will inhibit uracil nucleotide synthesis, and auxotrophic strains lacking this gene require the addition of uracil or uridine to grow.

[0068] 5′-Fluorouracid (5′-FOA) is an analogue of the precursor of uracil synthesis. Under the influence of ura5, it produces a cellularly toxic substance, therefore wild-type strains cannot grow in the presence of 5′-FOA. However, strains with the ura5 gene deletion cannot convert 5′-FOA into the toxic 5′-fluorouracil, exhibiting 5′-FOA resistance, and can thus be used for reverse selection. This phenotypic change caused by gene deletion or blockage can be directly observed in transformants, allowing for direct detection of gene editing efficiency.

[0069] To test the efficiency of the constructed single-base editing system in *Trichoderma tumefaciens*, an sgRNA sequence targeting the ura5 gene was designed using the online website CRISPOR. The sgRNA sequence targeting the ura5 gene is shown in SEQ ID NO. 7. The ura5 gene (GenBank number: PQ857720) was inactivated by introducing a C-to-T conversion 17 nucleotides upstream of the PAM sequence. This sequence was then ligated to the glycine promoter tRNA and the 6-T terminator of the sgRNA expression framework via fusion PCR, and assembled into a cytosine base editing plasmid to obtain the plasmid pTK-tRNA(Gly)-ura5-SDD7.

[0070] The plasmid was introduced into *Trichoderma truncatum* cells using a PEG-mediated method. Twenty-two transformants were randomly selected, and their genomic DNA was extracted. Using this DNA as a template, PCR amplification was performed on the target site region, and the amplified DNA fragments were sequenced. Sequencing results showed that three transformants had a C-to-T mutation 17 nucleotides upstream of the PAM sequence, with an editing efficiency of approximately 13.6%. Figure 5 ).

[0071] Example 4

[0072] Targeted editing of yak1

[0073] The serine protein kinase encoding gene yak1 (GenBank number: PQ781254) was selected as the research object. This gene has been shown to be involved in regulating vegetative growth, conidia formation, stress response, and inducing cellulase production. An sgRNA sequence targeting the yak1 gene was designed using the online website CRISPOR. The sgRNA base sequence targeting the yak1 gene is shown in SEQ ID NO.8. The sgRNA sequence targeting *Trichoderma thomsonii* yak1 was fused with the glycine promoter tRNA and a 6-T terminator of the sgRNA expression framework via fusion PCR, assembled into a cytosine base editing plasmid, obtaining the plasmid pTK-tRNA(Gly)-Yak1-SDD7, which was then transformed into *Trichoderma thomsonii*. We randomly selected 27 transformants, extracted their genomic DNA, and used this as a template to perform PCR amplification and sequencing of the target site region. The sequencing results showed that (…). Figure 6 22 transformants achieved base editing, with an editing efficiency of approximately 81.5%.

[0074] Example 5

[0075] Simultaneous editing of the ace1 and yak1 genes

[0076] We attempted to perform simultaneous dual-gene editing in *Trichoderma tumefaciens* using the mini-SDD7-CBE base editing system. We used ace1 (GenBank number: PQ781253) and yak1 as target genes. Using the online website CRISPOR, we designed an sgRNA sequence targeting the ace1 gene, as shown in SEQ ID NO. 9. The sgRNA sequence targeting *Trichoderma tumefaciens* ace1 was fused with the glycine promoter tRNA and a 6-T terminator of the sgRNA expression framework via fusion PCR, and then tandemly assembled with the sgRNA expression framework targeting *Trichoderma tumefaciens* yak1. Figure 7 ).

[0077] We used plasmid pTK-tRNA(Gly)-Yak1-SDD7 as the starting plasmid, linearized it with KpnI, and ligated it to an sgRNA expression framework to assemble the plasmid pTK-tRNA(Gly)-Ace1-Yak1-SDD7 based on the mini-SDD7-CBE editor. We introduced this plasmid into *Trichoderma tumefaciens*, randomly selected 30 transformants, and extracted their genomic DNA. Using these genomic DNAs as templates, we amplified the target site region and sequenced the amplified DNA fragments. Sequencing results showed that simultaneous editing of ace1 and yak1 was achieved in three transformants. Figure 8 The double gene editing efficiency was 10%. Among the 30 transformants, 19 transformedants underwent base editing at the expected site of yak1, and only 3 transformedants underwent base editing at the expected site of ace1. Furthermore, these 3 transformedants underwent simultaneous editing of two genes.

[0078] Example 6

[0079] Simultaneous editing of the pk1, ace1, and yak1 genes

[0080] To improve editing efficiency, we attempted simultaneous editing of multiple genes in *Trichoderma tumefaciens* using the mini-SDD7-CBE base editing system. We used pk1 (GenBank number: PQ845638), ace1, and yak1 as target genes. Using the online website CRISPOR, we designed an sgRNA sequence targeting the pk1 gene, as shown in SEQ ID NO. 10. The sgRNA sequence targeting *Trichoderma tumefaciens* pk1 was fused with the glycine promoter tRNA and the 6-T terminator of the sgRNA expression frame via fusion PCR, and then tandemly assembled with the sgRNA expression frames targeting yak1 and ace1. The plasmid pTK-tRNA(Gly)-Ace1-Yak1-SDD7 was linearized using KpnI and ligated to an sgRNA expression framework to assemble a mini-SDD7-CBE editor-based plasmid, pTK-tRNA(Gly)-pk1-Ace1-Yak1-SDD7. This plasmid was transformed into *Trichoderma cornuta*, and 30 transformants were randomly selected. Genomic DNA was extracted from each transformant. Using these genomic DNAs as templates, the target site regions were amplified, and the amplified DNA fragments were sequenced. Sequencing results showed that four transformants achieved simultaneous editing of the pk1, ace1, and yak1 genes. (Sequencing results are shown in the attached table.) Figure 9 The editing efficiency is 13%.

[0081] Example 7

[0082] Elimination of single-base editing plasmids

[0083] Transformants confirmed to be positive for editing were inoculated onto antibiotic-free plates and passaged continuously (48–72 h per generation, 3–5 rounds). Single colonies after passage were picked and inoculated onto plates with and without hygromycin, and the proportion of colonies losing their growth capacity was counted. Molecular validation: The hyg fragment was amplified by PCR using primers Hyg-VF / R. A negative result indicated that the vector had been lost. The results showed that the vector loss efficiency was approximately 88%. Figure 10 ).

[0084] References

[0085] [1] Liu R., Chen L., Jiang Y., Zhou Z. and Zou G. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR / Cas9system. Cell Discovery, 2015, 1: 15007

[0086] [2] Komor AC, Kim YB, Packer MS, Zuris JA and Liu DR. Programmable editing of a target base in genomic DNA without double-strandedDNA cleavage. Nature, 2016, 533(7603): 420-424

[0087] [3] Huang, J., Lin, Q., Fei, H., He, Z., Xu, H., Li, Y., Qu, K., Han, P., Gao, Q., Li, B., Liu, G., Zhang, L., Hu, J., Zhang, R., Zuo, E., Luo, Y., Ran, Y., Qiu, J.-L., Zhao, KT, & Gao, C. Discovery of deaminase functions by structure-based protein clustering. Cell, 2023, 186(15):3182-3195.e14.

[0088] [4] Duan Chengbao. Activity evaluation of commonly used promoters of Aspergillus niger and establishment of CRISPR / Cas9 gene editing system [D]. Beijing: Master's thesis of University of Chinese Academy of Sciences, 2020.

[0089] [5] Komor AC, Zhao KT, Packer MS, Gaudelli NM, WaterburyA. L., Koblan LW, Kim YB, Badran AH and Liu DR. Improved baseexcision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:Abase editors with higher efficiency and product purity. Science Advances, 2017, 3(8): eaao4774.

Claims

1. A cytosine base editing plasmid, characterized in that, Compared to the starting plasmid, the following sequence was inserted: Terminator Tcyc1; the base sequence of terminator Tcyc1 is shown in SEQ ID NO.5; Promoter PgpdA; the base sequence of the promoter PgpdA is shown in SEQ ID NO.1; mini-SDD7 deaminase; the amino acid sequence of the mini-SDD7 deaminase is shown in SEQ ID NO.2; The nCas9 (D10A) coding sequence; the amino acid sequence of the nCas9 (D10A) coding sequence is shown in SEQ ID NO.3; UGI coding sequence; the amino acid sequence of the UGI coding sequence is shown in SEQ ID NO.4; The starting plasmid is pUC19-AMA1-(P)Hyg.

2. A base editing system, characterized in that, Includes the cytosine base editing plasmid and sgRNA expression cassette as described in claim 1; The sgRNA expression cassette includes a glycine promoter tRNA, an sgRNA sequence with a target sequence designed for the edited gene, and a 6-T terminator. The base sequence of the glycine promoter tRNA is shown in SEQ ID NO.

6.

3. The base editing system according to claim 2, characterized in that, The target gene is ura5; The base sequence of the sgRNA sequence is shown in SEQ ID NO.

7.

4. The base editing system according to claim 2, characterized in that, The target gene is yak1; The base sequence of the sgRNA sequence is shown in SEQ ID NO.

8.

5. The base editing system according to claim 2, characterized in that, The target genes are ace1 and yak1; The base sequence of the sgRNA sequence of ace1 is shown in SEQ ID NO.9; The base sequence of the sgRNA of yak1 is shown in SEQ ID NO.8; The sgRNA expression cascade consists of two sgRNA expression cascades.

6. The base editing system according to claim 2, characterized in that, The target genes are pk1, ace1, and yak1; The base sequence of the sgRNA sequence of pk1 is shown in SEQ ID NO.10; The base sequence of the sgRNA sequence of ace1 is shown in SEQ ID NO.9; The base sequence of the sgRNA of yak1 is shown in SEQ ID NO.8; The sgRNA expression cascade consists of three sgRNA expression cascades.

7. The use of the base editing system according to any one of claims 2 to 6 in gene editing in Trichoderma syn.

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