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. 2026 Mar 1;17(3):718–734. doi: 10.1080/21501203.2026.2622835

CRISPR/Cas9 nickase-mediated cytosine base editing in Trichoderma koningiopsis

Yushi Fu a,b,*, Yuxia Duan a,*, Yongpeng Yao a, Yuanyuan Pan a, Zhaoqing Zeng a, Wenying Zhuang a, Gang Liu a,b,✉
PMCID: PMC13647404  PMID: 42851865

ABSTRACT

Base editors are regarded as reliable and precise editing tools due to their ability to efficiently and accurately modify the target genes without inducing double-strand breaks (DSBs). Notably, base editors offer distinct advantages in base editing, particularly in multiplex gene editing. However, application of base editors in the filamentous fungus Trichoderma remains underdeveloped. In this study, two cytosine base editors (CBE), designated as rAPOBEC1-CBE and mini-SDD7-CBE, respectively, were constructed in Trichoderma koningiopsis. Three targets related with cellulase production were chosen for testing editing efficiency of these two systems. The mini-SDD7-CBE system exhibited a maximum editing efficiency of 80%, with the editing window predominantly situated at positions 16 to 20 upstream of the PAM sequence in T. koningiopsis. Utilizing the mini-SDD7-CBE system, simultaneous editing of two and three genes was successfully executed. The overall editing efficiency was primarily influenced by the lowest editing efficiency of an individual single guide RNA (sgRNA). This study provides a convenient tool for the precise genome engineering of Trichoderma, thereby facilitating the extensive application of base editing techniques in filamentous fungi.

KEYWORDS: Cytosine base editor, Trichoderma koningiopsis, cellulase, xylanase, regulatory genes

GRAPHICAL ABSTRACT

graphic file with name TMYC_A_2622835_UF0001_OC.webp

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1. Introduction

The programmable genome editing technologies have profoundly transformed the fields of biological research and metabolic engineering in recent years. Of them, the CRISPR/Cas9 nickase-mediated base editors constitute a major innovation, facilitating precise single nucleotide alterations without necessitating double-strand breaks (DSBs) or donor DNA templates (Zhou et al. 2017; Anzalone et al. 2020). Canonical base editors generally consist of three critical components, including a catalytically inactive Cas9 variant (dCas9 or nCas9), base-modifying enzymes, and a uracil glycosylase inhibitor (UGI) (Billon et al. 2017; Kuscu et al. 2017). Base editors are further categorized into cytosine base editors (CBEs) and adenine base editors (ABEs), distinguished by the base-modifying enzymes (Rees and Liu 2018). The CBE system operates through the fusion of cytosine nucleoside deaminase with either nCas9 or dCas9 to form effector proteins. Utilizing single-guide RNA (sgRNA) for precise targeting, this system directs the modification of specific DNA sequences by catalyzing the deamination of cytosine nucleosides (C) into uracil nucleosides (U) within a designated region of the target site. Through subsequent DNA replication and repair mechanisms, these uracil nucleosides are converted into thymidine nucleosides (T), finally completing the conversion of C-to-T within the target DNA sequence. Concurrently, UGI is employed to inhibit unintended base excision (Komor et al. 2016).

Since the first report of CBE in 2016, substantial improvements have been made to enhance editing efficiency and specificity. The BE3 comprising the rat APOBEC1, nCas9 (D10A), and UGI demonstrated an efficient C-to-T conversion in mammalian cells (Yan et al. 2023). BE4 was subsequently developed by optimizing the linker lengths and incorporating an additional UGI domain, showing improved editing precision (Koblan et al. 2018). BE4max, featuring codon optimization and modified nuclear localization signals (NLS), achieved even higher editing efficiencies (Komor et al. 2017). Parallel efforts led to the development of Target-AID, which utilizes the PmCDA1 deaminase from sea lamprey (Ma et al. 2016). The evolved deaminases such as evoCDA1 and evoAPOBEC1 have been engineered to overcome sequence context limitations, enabling efficient editing of cytosines in previously restricted contexts, particularly in GC-rich regions (Thuronyi et al. 2019). Recently, the SDD7 deaminase was selected by structure-based protein prediction and has been documented to exhibit an editing efficiency 3.5 times higher than that of the rAPOBEC1 deaminase in rice, while also maintaining high editing activity in both human cells and soybean (Huang et al. 2023), demonstrating its potential for cross-species application. Notably, SDD7 does not show sequence context preference in contrast to rAPOBEC1, which exhibits a marked preference for 5’-TC and 5’-CC bases.

Due to their capacity to precisely substitute a single nucleotide without inducing DSBs, base editors have superior potential for multigene editing compared to the traditional CRISPR/Cas9 system. Moreover, base editors exhibit an enhanced performance in minimizing off-target effects (Doman et al. 2020). However, application of the CRISPR/Cas9 nickase-mediated base editors for multigene editing in filamentous fungi remains underdeveloped and requires further advancement.

The genus Trichoderma is well known for producing cellulolytic and hemicellulolytic enzymes to degrade lignocellulolytic enzymes and has been extensively studied (Strakowska et al. 2014; Zhao et al. 2025). Besides Trichoderma reesei, which is the industrial producer of cellulolytic and hemicellulolytic enzymes, other Trichoderma species including Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, and Trichoderma viride, also have the strong ability to degrade crystalline cellulosic substrates (Gusakov 2011). Trichoderma koningiopsis emerges as a promising candidate for industrial enzyme production due to its exceptional cellulases and hemicellulases-producing capabilities (Li and Zhuang 2022; Zhang et al. 2025). However, the difficulty of genetically manipulating Trichoderma significantly hampers its targeted and rational genetic modification. Recently, the CRISPR/Cas9 system has been applied in T. reesei (Liu et al. 2015), facilitating efficient gene deletions and insertions. Nonetheless, the application of base editing in Trichoderma remains largely underexplored.

In this study, two cytosine base editors designated as rAPOBEC1-CBE and mini-SDD7-CBE were developed for genome editing of T. koningiopsis. These editors were successfully utilized to achieve targeted gene inactivation by converting specific codons (CAA, CAG, or CGG) into stop codons (TAA, TAG, or TGA). Notably, the mini-SDD7-CBE system demonstrated a maximum editing efficiency of 80%. Utilizing the mini-SDD7-CBE system, we successfully accomplished concurrent editing of both two and three genes. Furthermore, the overall efficiency of multigene editing is predominantly determined by the single-guide RNA (sgRNA) exhibiting the lowest editing efficiency. Our work provides a new tool for precise genome engineering in Trichoderma and contributes to the broader application of base editing in filamentous fungi.

2. Materials and methods

2.1. Strains, media, and growth conditions

The T. koningiopsis strain was cultivated on potato dextrose agar (PDA) at 28 °C for 5–7 days to produce conidia. Antibiotics were used as necessary to select the transformants. For liquid fermentation and crude enzyme preparation, strains were grown on PDA for 3 days. Four 1 cm2 mycelium pieces were inoculated into 100 mL of the PDB liquid medium with glass beads and incubated at 28 °C and 220 r/min for 48 h as seed fluid. Then, 10 mL of inoculum was transferred into 100 mL of the cellulase production medium and cultured at 28 °C and 180 r/min. After being cultured for 7 days, the fermentation broth was collected, respectively, and centrifuged at 4 °C and 10,000 r/min for 10 min, and the supernatant was collected as crude enzyme fluid and stored at 4 °C for enzymatic analysis. The cellulase production medium was composed as follows: 2% crystalline cellulose (Avicel), 2% bran, 1% peptone, 1.5% KH2PO4, 0.5% (NH4)2SO4, 0.06% MgSO4, 0.08% CaCl2, 0.08% Urea, 0.00016% MnSO4‧H2O, 0.00005% FeSO4‧7H2O, 0.00014% ZnSO4‧7H2O, 0.00002% CoCl2, pH 4.8. For vector manipulation and propagation, Escherichia coli DH5α (Tsingke, Beijing) was cultured at 37 °C in Luria-Bertani broth supplemented with ampicillin (100 μg/mL).

2.2. Construction of the plasmid used for base editing

The coding sequences of the effector elements were optimized for Trichoderma codons and synthesized by GenScript Biotech Corp (Nanjing, China). As an illustrative example, the construction process of the base editing plasmid pUC19-AMA1-Hyg-rAPOBEC1-CBE was described, wherein the fusion protein encoding genes were expressed under control of the gpdA promoter (PgpdA) from Aspergillus nidulans (Chen et al. 2019). Initially, the AMA1 replicator from A. nidulans and the selective marker hyg (GenBank U40398.1) were cloned into the plasmid pUC19, generating the plasmid pUC19-AMA1-Hyg. Subsequently, the primers PgpdA-F/PgpdA-R, rApo-F/rApo-R, Cas-ugi-F/Cas-ugi-R, and Tcyc1-F/Tcyc1-R were employed to amplify the gpdA promoter (PgpdA), rAPOBEC1-XTEN, nCas9-SGGS-UGI-NLS, and cyc1 terminator (Tcyc1), respectively. These amplified fragments were then assembled into pUC19-AMA1-Hyg by using the SE Seamless Cloning and Assembly Kit (Zomanbio, Beijing), generating the plasmid pUC19-AMA1-Hyg-rAPOBEC1-CBE. The construction process of the plasmid pUC19-AMA1-Hyg-mini-SDD7-CBE was constructed following the same strategy as that used for pUC19-AMA1-Hyg-rAPOBEC1-CBE. Specifically, the primers PgpdA2-F/PgpdA2-R, sdd7-F/sdd7-R, Cas-F/Cas-R, ugi-F/ugi-R, and Tcyc1-F/Tcyc1-R were employed to amplify the gpdA promoter (PgpdA), NLS-mini-SDD7-XTEN, nCas9-NLS-SGGS, UGI-NLS, and cyc terminator (Tcyc), respectively. These amplified fragments were subsequently assembled into the pUC19-AMA1-Hyg backbone using the SE Seamless Cloning and Assembly Kit, resulting in pUC19-AMA1-Hyg- miniSDD7-CBE.

The sgRNA cassette comprised three components including the promoter responsible for driving sgRNA expression, the specific sgRNAs targeting the gene of interest, and the terminator with six thymine residues to terminate sgRNA expression. The glycine tRNA (GenBase number: C_AA104155.1, https://ngdc.cncb.ac.cn/genbase) from T. koningiopsis was selected to drive sgRNA expression. To identify the specific sgRNAs targeting the genes ura5 (GenBank number: PQ857720), ace1 (GenBank number: PQ781253), yak1 (GenBank number: PQ781254), and pk1 (GenBank number: PQ845638), all potential sgRNA target sites within the genome of T. koningiopsis were determined using the CRISPOR tool (http://crispor.tefor.net/crispor.py) (Concordet and Haeussler 2018), and sgRNA target sites with high scores were chosen. The sgRNA cassette was integrated into the KpnI site of pUC19-AMA1-Hyg-rAPOBEC1-CBE or pUC19-AMA1-Hyg-mini-SDD7-CBE via the SE Seamless Cloning and Assembly Kit to generate the final base editing plasmids.

2.3. Base editing in T. koningiopsis

The PEG-mediated transformation of T. koningiopsis protoplasts was performed as described previously (Penttilä et al. 1987). Five micrograms of plasmid DNA were transformed into 1 × 107 T. koningiopsis protoplasts. Colonies grown on MM at 28 °C for 7 days were screened using 100 μg/mL hygromycin B (Invivogene, USA), followed by sequential identification via PCR analysis and DNA sequencing. Genomic DNA was extracted from the collected mycelia after cultured for 48 h in PDB medium and used as template. Editing efficiency was calculated based on the DNA sequencing results. The mutants were subjected to 3–4 consecutive rounds of subculture to remove the editing plasmids.

2.4. Off-target analysis

Off-target analysis was performed as follows. Potential off-target sites for the sgRNA were predicted using CasOFFinder (http://www.rgenome.net/cas-offinder/). The top 25 candidate loci were selected, and ~500 bp upstream and downstream regions were PCRamplified from genomic DNA of the edited strain and the wildtype strain. Amplicons were subjected to Sanger sequencing, and chromatograms were analyzed using TIDE (https://apps.datacurators.nl/tide/) to quantify indel frequencies. Indels were considered as potential off-target events only when the associated p value was < 0.001.

2.5. Enzyme activity measurements

The activity of carboxymethylcellulase was assessed using the dinitrosalicylic acid (DNS) method with carboxymethyl cellulose sodium salt (CMC-Na) serving as the substrate (Ghose 1987). Similarly, the activity of endo-1,4-β-xylanase was evaluated using the DNS method with xylan (Ruitaibio, Beijing) as the substrate (König et al. 2002). An international unit (IU) was defined as the amount of enzyme required to catalyze the formation of 1 μmol of reducing sugar per minute under the conditions of 50 °C and pH 4.8, per milliliter of liquid enzyme. All estimations were conducted in triplicate assays.

The total protein concentration in the fermentation broth was determined using the BCA assay. According to the manufacturer’s instructions, the fermentation supernatant was mixed with the BCA working solution and incubated at 37 °C for 30 min. The absorbance was measured at 562 nm, and the protein concentration was calculated based on a standard curve, using the absorbance of uninoculated fermentation supernatant as the blank.

2.6. Total RNA extraction and quantitative RT-PCR

Total RNA was extracted from the fungal mycelia by using Ultrapure RNA Kit (Cwbio, China), following the protocol provided by the manufacturer. The isolated RNA was quantified by a NanoDrop LiTE Spectrophotometer (Thermo Scientific, United States). Subsequently, 1 µg of total RNA was reverse transcribed into the complementary DNA (cDNA) by using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China), in accordance with the manufacturer’s instruction. The optimized quantitative PCR (qPCR) reactions comprised 2 μL of cDNA template, 0.4 μmol/L of each primer, and 2× ChamQ Universal SYBR qPCR Master Mix, resulting in a final volume of 20 μL. The primers used for RT-qPCR are detailed in Table S2. Real-time reverse transcription PCR was performed by using the BioRad CFX96 Touch Real-Time PCR Detection System, with each sample being tested in triplicates. For qPCR, actin serves as the internal reference gene. Relative expression was calculated as 2−ΔΔCt, where ΔCt = Ct(target) – Ct(actin), and WT was used as the calibrator (fold change = 1). Thus, all mutant values represent expression normalized to actin and then expressed relative to WT.

2.7. Statistical analyses

The statistical significance of differences between the two conditions was evaluated utilizing a two-tailed Student’s t-test.

3. Results

3.1. Construction of the cytosine base-editing system in T. koningiopsis

To carry out the genome editing of T. koningiopsis, we constructed two nCas9 (D10A)-based genome-editing systems designated rAPOBEC1-CBE and mini-SDD7-CBE, respectively (Figure 1a,b) (Komor et al. 2016; Huang et al. 2023). The gpdA promoter from A. nidulans was utilized in rAPOBEC1-CBE to facilitate expressing the recombinant gene encoding the fusion protein comprising a deaminase (rat APOBEC1), Cas9 nickase (Streptococcus pyogenes Cas9D10A), and UGI from the Bacillus subtilis bacteriophage PBS2. The rAPOBEC1 deaminase was linked to the N-terminus of nCas9 via a 16-aa XTEN linker, and UGI was connected to the C-terminus of nCas9 via the SGGS linker. To enhance the localization of the fusion protein, an SV40 NLS was conjugated to the C-terminus of the fusion construct. The glycine tRNA(GCC) from T. koningiopsis was selected as an effective transcriptional control element for sgRNA expression (Yuan and Gao 2022). In mini-SDD7-CBE, the rAPOBEC1 deaminase was substituted with mini-SDD7 which was engineered by excising redundant sequences from the Sdd7 deaminase originally derived from Kribbella voronezhensis (Huang et al. 2023). The SV40 NLS was integrated between nCas9 and UGI, and conjugated to the N-terminus of the fusion protein.

Figure 1.

Figure 1.

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Schematic representation of the base-editor structure. (a) Schematic diagram of rAPOBEC1-CBE. (b) Schematic diagram of mini-SDD7-CBE with sgRNA cassette. Hyg, the hygromycin B resistance gene. AMA1, the autonomous replication sequence. PgpdA and Tcyc1 indicate the gpdA promoter and the cyc1 terminator respectively. NLS, the nuclear localization sequence. rAPOBEC1, the rat cytosine deaminase. mini-SDD7, the bacterial cytosine deaminase from Kribbella voronezhensis. XTEN, the linker sequence. nCas9(D10A), Cas9 nickase. SGGS, the linker sequence. UGI, the uracil DNA glycosylase inhibitor. Amp, the ampicillin resistance gene. Ori, origin of replication. PtRNAGly indicates the glycine tRNA from Trichoderma koningiopsis; crRNA indicates CRISPR RNA; tracrRNA indicates trans-activating crRNA. Tpoly Tx8 denotes polythymidine, specifically utilizing eight thymine bases as a terminator to halt the transcription of sgRNA. Each base editor for target gene was linked to the KpnI site of plasmid. (c) The growth of OE-rAPOBEC1-CBE, OE-mini-SDD7-CBE, and T. koningiopsis (WT) on the minimal medium after 2 days of culture. OE-rAPOBEC1-CBE to the strain with the rAPOBEC1-CBE system (without the sgRNA cassette), OE-mini-SDD7-CBE to the strain with the mini-SDD7-CBE system (without the sgRNA cassette). (d) The growth diameter of OE-rAPOBEC1-CBE, OE-mini-SDD7-CBE, and T. koningiopsis (WT) on the minimal medium after 2 days of culture. Dates were presented as the Mean ± SD (n = 3). ns, not significant.

3.2. Assessment of the editing efficiency of the base editors in T. koningiopsis

To evaluate effects of the rAPOBEC1-CBE and mini-SDD7-CBE on the genome editing of T. koningiopsis, the corresponding transformants carrying each base editor were obtained and designated as OE-rAPOBEC1-CBE and OE-mini-SDD7-CBE, respectively. Analysis of the growth diameters indicated that rAPOBEC1-CBE and mini-SDD7-CBE did not affect the growth of T. koningiopsis (Figure 1c,d).

To test the editing efficiency of these two CBE systems in T. koningiopsis, the orotidine 5’-phosphate decarboxylase gene ura5 was initially selected as the editing target. Ura5 is responsible for converting 5′-fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil (5-FU) through the intermediate 5-fluorouridylic acid (5-FUMP) (Melin et al. 2008). Deletion of ura5 resulted in partial resistance of Trichoderma to 5-FOA (Liu et al. 2015). Therefore, we designed an sgRNA sequence (5′-ATCCAGGCGCAGCAGGAGTC-3′) for inactivating the ura5 by introducing a nonsense mutation via a C to T transition at −17 nt from the PAM sequence (Figure 2a).

Figure 2.

Figure 2.

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Comparison of editing efficiency between rAPOBEC1-CBE and mini-SDD7-CBE. (a) Sequence alignment of the ura5 locus edited by rAPOBEC1-CBE or mini-SDD7-CBE. (b) Sequence alignment of the yak1 locus edited by rAPOBEC1-CBE or mini-SDD7-CBE. The PAM sequence is highlighted in green. The expected editing site is shown by the nucleotide number relative to the PAM sequence and shaded in grey. The translated amino acid sequences are indicated under each nucleotide sequence. Mutated bases and amino acids are highlighted in red. The site-specific editing efficiencies for each editor are annotated in the figures as percentages.

The 5’FOA-resistant transformants were selected, and the mutations in the target region of ura5 were confirmed through DNA sequencing. The editing efficiency of rAPOBEC1-CBE was 11.1% (5 out of 45 transformants), while that of mini-SDD7-CBE was 13.6% (3 out of 22 transformants). The results revealed that all five transformants edited with rAPOBEC1-CBE exhibited a C to T conversion at −17 nt from the PAM sequence. However, three transformants withmini-SDD7-CBE displayed a C to T mutation at −18 to −17 nt from the PAM sequence (Figure 2a).

To further evaluate the editing efficiency of these two CBE systems, the serine protein kinase gene yak1 was selected as the target. The yak1 gene is involved in regulation of vegetative growth, conidiation, stress response, and induced cellulase production in Trichoderma reesei (Lv et al. 2015). The sequence of the protospacer region targeting yak1 is presented in Figure 2b. We constructed the expression cassettes by framing rAPOBEC1-CBE and mini-SDD7-CBE with sgRNA driven by a glycine tRNA (GenBase number: C_AA104155.1). These constructs were subsequently introduced into T. koningiopsis. Transformants were selected at random, and the target site region was sequenced. The results indicated that the yak1 editing efficiency mediated by rAPOBEC1-CBE was 12.5% (3 out of 24). Two transformants demonstrated a C to T mutation located 14 nt upstream of the PAM sequence, whereas one transformant exhibited a C to T mutation at 7 nt upstream of the PAM sequence. The latter mutation did not result in the inactivation of yak1. Unexpectedly, the mini-SDD7-CBE-mediated editing efficiency of yak1 was remarkably high up to 81.5% (22 out of 27), indicating the mini-SDD7-CBE system was superior to that of the rAPOBEC1-CBE. The editing sites of yak1 were predominantly concentrated at −17 nt from the PAM sequence, with additional mutations detected at positions −9, −11, and −14 from the PAM sequence (Figure 2b). This observation was consistent with the reported editing efficiency of the mini-SDD7 deaminase, which is effective at positions ranging from 10 to 20 nt upstream of the PAM sequence in human cells (Huang et al. 2023). Concurrently, it was noted that some transformants achieved the base conversion of C to A/G or base deletions. We hypothesize that these unintended mutations may result from the mini-SDD7-CBE-mediated deamination occurring at multiple loci, thereby activating various DNA repair mechanisms that lead to mis-repair and fragment rearrangement. The failure of the rAPOBEC1-CBE system to achieve the anticipated mutation at position −17 upstream of the PAM site may be attributed to the specific motif editing preferences of the rAPOBEC1 deaminase. The presence of a 5’-GC sequence likely inhibited the deaminase’s expected activity. Consequently, considering both editing efficiency and mode, we ultimately selected the mini-SDD7-CBE system for further investigation.

Utilizing the Cas-OFFinder local platform, we identified 25 potential off-target sites within the genome for the sgRNA targeting yak1, which exhibited the highest editing activity with mini-SDD7-CBE. For each of these candidate sites, DNA fragments approximately 500 base pairs in length, flanking the predicted protospacer adjacent motif, were amplified by PCR from both the edited and wild-type strains and subsequently subjected to Sanger sequencing. The resulting chromatograms were analyzed using the TIDE algorithm to quantify the insertion-deletion (indel) frequencies at each locus (Table 1). Across all 25 candidate off-target sites, the “overall efficiency” (quantified indel frequency) values determined by TIDE ranged from 0% to 4.4% (Figures S1–S3). According to the TIDE quality criteria, such low-level signals with non-significant p values are considered indistinguishable from background noise. Thus, no statistically supported off-target editing events were detected at any of the predicted sites, indicating that the mini-SDD7-CBE system exhibits no appreciable off-target activity at the computationally predicted loci under our experimental conditions.

Table 1.

TIDE-based off-target analysis for 25 predicted sites during targeting yak1 with sgRNA in mini-SDD7-CBE.

Off-target ID Mismatches to sgRNA Overall efficiency
(% indels)		 p value Significant off-target
OFT-1 5 0.70 ≥0.001 No
OFT-2 5 0 ≥0.001 No
OFT-3 5 0.80 ≥0.001 No
OFT-4 5 0.80 ≥0.001 No
OFT-5 4 0.80 ≥0.001 No
OFT-6 5 1.20 ≥0.001 No
OFT-7 5 1.70 ≥0.001 No
OFT-8 5 1.40 ≥0.001 No
OFT-9 5 2 ≥0.001 No
OFT-10 5 1.70 ≥0.001 No
OFT-11 5 0.80 ≥0.001 No
OFT-12 5 1.60 ≥0.001 No
OFT-13 5 2.30 ≥0.001 No
OFT-14 5 1.10 ≥0.001 No
OFT-15 5 0.70 ≥0.001 No
OFT-16 5 1.30 ≥0.001 No
OFT-17 5 1.10 ≥0.001 No
OFT-18 5 1.60 ≥0.001 No
OFT-19 5 4.40 ≥0.001 No
OFT-20 5 2.60 ≥0.001 No
OFT-21 5 1.70 ≥0.001 No
OFT-22 5 0.90 ≥0.001 No
OFT-23 5 1.90 ≥0.001 No
OFT-24 5 0.40 ≥0.001 No
OFT-25 5 1 ≥0.001 No
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3.3. Expansion of mini-SDD7-CBE in editing the ace1 and pk1 genes

To further validate the editing efficiency of mini-SDD7-CBE, we selected two additional genes involved in the regulation of cellulase production as the targets. The ace1 gene encodes a transcriptional repressor of cellulase production in Trichoderma (Saloheimo et al. 2000). The pk1 gene was identified through KEGG annotation as a potential mitogen-activated protein kinase coding gene; its product exhibits 97% similarity to TMK2 which is implicated in maintaining cell wall integrity in T. reesei (Wang et al. 2014). The sgRNA expression cassettes for targeting ace1 and pk1 were designed and individually assembled into pUC-AMA1-Hyg-mini-SDD7-CBE. The resulting plasmids were subsequently transferred into T. koningiopsis. The mini-SDD7-CBE mediated base editing with efficiency of 15% for ace1 and 10% for pk1. Among the transformants with the ace1 editing, three exhibited a C-to-T mutation at −16 nt upstream of the PAM sequence (Figure 3a). Similarly, among the transformants with the pk1 editing, three exhibited C-to-T mutations at −17 and −11 nt upstream of the PAM sequence (Figure 3a). By analyzing the editing sites generated by mini-SDD7-CBE in ace1 and pk1, we observed that its editing window in T. koningiopsis predominantly spans positions −16 to −20 nt upstream of the PAM sequence. This CBE system exhibited a flexible editing window. However, its editing efficiency was significantly influenced by the compatibility of the sgRNA. Compared to ura5, ace1, and pk1, the mini-SDD7-CBE system demonstrated a significantly higher base editing efficiency for yak1. This result indicated the critical influence of sgRNA selection and chromatin accessibility at target sites on the overall efficiency of the base editor (Shalem et al. 2015).

Figure 3.

Figure 3.

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Evaluation of mini-SDD7-CBE efficiency in single and multiplex gene editing. (a) Sequence alignment of the ace1 or pk1 locus modified by mini-SDD7-CBE. The PAM sequence is highlighted in green. The expected editing site is shown by the nucleotide number relative to the PAM sequence and shaded in grey. The translated amino acid sequences are indicated under each nucleotide sequence. Mutated bases and amino acids are highlighted in red. The site-specific editing efficiencies for each editor are annotated in the figures as percentages. (b) Schematic diagram of plasmid pTK-tRNA(Gly)-Ace1-Yak1-SDD7. Hyg, the hygromycin B resistance gene. AMA1, the autonomous replication sequence. PgpdA and Tcyc1 indicate the gpdA promoter and the cyc1 terminator respectively. NLS, the nuclear localization sequence. rAPOBEC1, the rat cytosine deaminase. mini-SDD7, the bacterial cytosine deaminase from Kribbella voronezhensis. XTEN, the linker sequence. nCas9(D10A), Cas9 nickase. SGGS, the linker sequence. UGI, the uracil DNA glycosylase inhibitor. Amp, the ampicillin resistance gene. Ori, origin of replication. PtRNAGly indicates the glycine tRNA from Trichoderma koningiopsis. yak1 gRNA: sgRNA targeting the yak1 gene and the tracrRNA. ace1 gRNA: sgRNA targeting the ace1 gene and the tracrRNA. Tpoly Tx8 denotes polythymidine, specifically utilizing eight thymine bases as a terminator to halt the transcription of sgRNA. Each base editor for target gene was linked to the KpnI site of plasmid. (c) Schematic diagram of plasmid pTK-tRNA(Gly)-Pk1-Ace1-Yak1-SDD7. Hyg, the hygromycin B resistance gene. AMA1, the autonomous replication sequence. PgpdA and Tcyc1 indicate the gpdA promoter and the cyc1 terminator respectively. NLS, the nuclear localization sequence. rAPOBEC1, the rat cytosine deaminase. mini-SDD7, the bacterial cytosine deaminase from Kribbella voronezhensis. XTEN, the linker sequence. nCas9(D10A), Cas9 nickase. SGGS, the linker sequence. UGI, the uracil DNA glycosylase inhibitor. Amp, the ampicillin resistance gene. Ori, origin of replication. PtRNAGly indicates the glycine tRNA from T. koningiopsis. yak1 gRNA: sgRNA targeting the yak1 gene and the tracrRNA. ace1 gRNA: sgRNA targeting the ace1 gene and the tracrRNA. pk1 gRNA: sgRNA targeting the pk1 gene and the tracrRNA. Tpoly Tx8 denotes polythymidine, specifically utilizing eight thymine bases as a terminator to halt the transcription of sgRNA. Tpoly Tx8 denotes polythymidine, specifically utilizing eight thymine bases as a terminator to halt the transcription of sgRNA. Each base editor for target gene was linked to the KpnI site of plasmid.

3.4. Mini-SDD7-CBE-mediated multiplex genome editing in T. koningiopsis

Leveraging the mini-SDD7-CBE system’s capability for single-site editing, we assessed its potential for multiplex gene editing in T. koningiopsis. Our objective was to simultaneously introduce premature stop codons into the ace1 and yak1 genes using this system. We selected ace1 and yak1 as target genes and employed a glycine tRNA promoter to drive the expression of sgRNAs. The sgRNA expression constructs were arranged in a tandem configuration to facilitate this process (Figure 3b). These constructs were subsequently integrated into the mini-SDD7-CBE expression plasmid, generating pTK-tRNA(Gly)-Ace1-Yak1-SDD7. This plasmid was introduced into T. koningiopsis. Among 30 randomly selected transformants, 3 exhibited successful simultaneous editing of ace1 and yak1, yielding a double gene editing efficiency of 10% (Table 2). Furthermore, among the transformants, 16 exhibited only base editing at the targeted yak1 locus. The editing efficiency for yak1 was approximately 63%, while the editing efficiency for ace1 was only around 10%. In addition to its application in single-gene editing, the mini-SDD7-CBE system demonstrated high editing efficiency for the yak1 gene in dual-gene editing scenarios. Subsequently, the mini-SDD7-CBE system was utilized for simultaneously editing three genes including pk1, ace1, and yak1. Employing a similar methodology, the tRNA (Gly) promoter was used to drive the expression of the sgRNA targeting pk1, ace1, and yak1, respectively, and three sets of sgRNA expression cassettes were assembled, resulting in pTK-tRNA (Gly)-Pk1-Ace1-Yak1-SDD7 (Figure 3c) which was subsequently introduced into T. koningiopsis. The results demonstrated that 4 of 30 randomly selected transformants successfully achieved the simultaneous editing of pk1, ace1, and yak1, yielding an editing efficiency of 13% (Table 2). A notable decrease in per-locus efficiencies was observed for the yak1 locus in multiplex configurations, probably due to competition among multiple sgRNAs for the Cas-deaminase complex as previously reported (Citorik et al. 2014), as well as locus-specific accessibility or repair dynamics (Isaac et al. 2016). As a result, the overall outcome of multiplex editing tends to reflect the efficiency of the least editable target (ace1/pk1), rather than suggesting an inherent reduction in the editability of yak1.

Table 2.

Statistical analysis of editing efficiency in the mini-SDD7-CBE system.

Target Number of mutants Number of transformants C to T frequency (%) Genotype of mutations
ura5 3 22 13.6 C−18C−17 >T−18T−17 (3)
yak1 22 27 81.5 C−17 >T−17 (5);
C−17C−14 >T−17T−14 (3);
C−17C−14C−11 >T−17T−14 T−11 (1);
C−17C−9 >T−17T−9 (3);
C−17C−14 >T−17A−14 (1);
C−17 >G−17 (7);
C−17 >T−17 and after C−11 base deletions (2)
ace1 3 20 15 C−16 >T−16 (3)
pk1 3 30 10 C−17C−11 >T−17T−11 (3)
yak1 & ace1 3 30 10 yak1: C−17 >T−17, ace1: C−16C−13 >T−16T−13 (1);
yak1: C−17C−9 >T−17T−9, ace1: C−16C−13C−11 >T−16T−13 T−11 (1);
yak1: C−17 >T−17, ace1: C−13 >T−13 (1)
yak1 & ace1 & pk1 4 30 13 yak1: C−17C−11 >T−17T−11, ace1: C−16 >T−16, pk1: C−17 >T−17 (1);
yak1: C−17 >T−17, ace1: C−18 >T−18, pk1: C−17 C−11 >T−17T−11 (1);
yak1: C−17 >T−17, ace1: C−16 >T−16, pk1: C−17 >T−17 (1);
yak1: C−17 >T−17, ace1: C−19 >T−19, pk1: C−17 >T−17 (1)
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The notation “C−18C−17 >T−18T−17 (3)” in the “Genotype of mutations” column signifies that, in three transformants, the cytosine bases located at positions −18 and −17 upstream of the PAM site were substituted with thymine bases. The numeral in parentheses, “(3)” specifies the quantity of transformants.

3.5. Acquisition of high-yielding xylanase strains through multiplex editing with the mini-SDD7-CBE system

As previously outlined, three genes associated with cellulase production in T. koningiopsis were genetically engineered by the mini-SDD7-CBE system, resulting in the generation of mutants including single, double, and triple gene disruption variants. Carboxymethylcellulase and xylanase activities were tested in both the wild-type strain (WT) and the mutants (Δyak1, Δace1, Δpk1, Δyak1Δace1, and Δyak1Δace1Δpk1, respectively). As shown in Figure 4a, Δyak1 produced more carboxymethylcellulase and xylanase than WT. The productions of carboxymethylcellulase and xylanase were 1.6 times and 1.3 times higher than that of WT, respectively. Δpk1, as well as Δyak1Δace1Δpk1, exhibited a substantial enhancement in xylanase production following a three-day fermentation period (Figure 4b). Notably, the xylanase production in Δpk1 increased by more than twofold compared to WT. In contrast, deletion of ace1 did not lead to a significant change in CMCase or xylanase activity. To minimize potential bias from differences in biomass and secretory capacity, cellulase and hemicellulase activities were also normalized to the total extracellular protein concentration in the fermentation supernatant. The specific activities (per mg protein) showed trends consistent with the volumetric activities, indicating that the enhanced cellulase production in Δyak1, the limited effect of Δace1, and the opposite effects of Δpk1 on cellulase versus hemicellulase production are not solely attributable to differences in culture density (Figure S4).

Figure 4.

Figure 4.

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Evaluation of cellulase and xylanase activities and analysis of encoding gene transcription in cellulase expression regulatory factor-deficient strains. (a) Assessment of carboxymethylcellulase activity in strains with inactivated cellulase expression regulatory factors. (b) Assessment of xylanase activity in strains with inactivated cellulase expression regulatory factors. (c–g) Quantitative reverse transcription PCR (RT-PCR) was employed to analyze the relative transcriptional levels of cellulase-encoding genes, including cbh1, cbh2, eg1, eg2, and bgl1, following a five-day fermentation period in various strains with inactivated cellulase transcriptional regulators. (h–j) Similarly, quantitative RT-PCR was used to evaluate the relative transcriptional levels of xylanase-encoding genes, including xyn1, xyn2, and bxl, after five days fermentation in different strains with inactivated cellulase transcriptional regulators. The actin served as the internal reference gene. Relative transcriptional level was calculated as 2−ΔΔCt, where ΔCt = Ct(target) − Ct(actin), and WT was used as the calibrator (fold change = 1). Thus, all mutant values represent expression normalized to actin and then expressed relative to WT. Error bars represent standard deviations derived from three independent assays. The Δyak1 designation refers to the yak1-inactivated strain, Δace1 to the ace1-inactivated strain, Δpk1 to the pk1-inactivated strain, Δyak1Δace1 to the strain with both yak1 and ace1 inactivated, and Δyak1Δace1Δpk1 to the strain with yak1, ace1, and pk1 all inactivated. Statistical significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

We analyzed the transcriptional levels of the genes encoding cellulases, specifically cbh1, cbh2, eg1, and eg2 (Luo et al. 2020), as well as bgl1 (Mach et al. 1995) and hemicellulases, including xyn1 and xyn2 (Zeilinger et al. 1996), and bxl (Akel et al. 2009). As illustrated in the figure (Figure 4c–j), inactivation of the pk1 gene resulted in a significant reduction in the transcription levels of cbh1, cbh2, and eg2 compared to the WT, while the transcription level of xyn1, xyn2, and bxl were increased approximately by tenfold after 5 days. The transcriptional levels of these genes were also assessed on the third and seventh days of fermentation, revealing similar patterns (Figures S5, S6). Consequently, we hypothesized that pk1 encoding a potential mitogen-activated protein kinase played a direct or indirect role in regulating the expression of cellulase-related genes and could have a broader impact on the organism’s physiological activities in T. koningiopsis.

Analysis of colony diameters of the aforementioned strains on basal media supplemented with various carbon sources revealed that Δpk1 and Δyak1Δace1Δpk1 exhibited significantly reduced growth compared to WT. Additionally, the growth rate of the Δyak1 strain was significantly lower than that of the WT only under glucose-based culture conditions. In contrast, no substantial growth defects or abnormal colony morphologies were observed in the other genetically edited strains (Figure 5b). In summary, utilizing the mini-SDD7-CBE editing system, we successfully obtained the cellulase high-producing strain Δyak1 and the xylanase high-producing strain Δyak1Δace1Δpk1.

Figure 5.

Figure 5.

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Examination of growth patterns in the strains deficient in cellulase transcriptional regulators. (a) Analysis of growth of the cellulase transcriptional regulator-deficient strains in various carbon source media. (b) Comparative assessment of fungal colony diameters in the cellulase transcriptional regulator-deficient strains cultured in media with different carbon sources. Dates were presented as the Mean ± SD (n = 3). The Δyak1 designation refers to the yak1-inactivated strain, Δace1 to the ace1-inactivated strain, Δpk1 to the pk1-inactivated strain, Δyak1Δace1 to the strain with both yak1 and ace1 inactivated, and Δyak1Δace1Δpk1 to the strain with yak1, ace1, and pk1 all inactivated. Statistical significance is denoted as follows: ****p < 0.0001.

4. Discussion

CBEs are the dependable gene editing tools for precisely and efficiently modifying the target genes without inducing double-strand breaks (DSBs). Currently, through the incorporation of UGIs (Komor et al. 2017), integration of other functional molecules such as NLS (Koblan et al. 2018) and DNA repair-related proteins (Zhang et al. 2020), as well as the modification or substitution of cytosine deaminase (Huang et al. 2019b), a variety of CBE systems have been developed. These systems, including BE3, BE4, tBEs, and Target-AID, exhibit high editing efficiency and reduced off-target effects (Wang et al. 2017; Zhao et al. 2019; Wang et al. 2021). Of them, the mini-SDD7-based CBE exhibits high editing efficiency (Huang et al. 2023). The mini-SDD7 based CBE also demonstrated cross-species applicability and low toxicity. The CBE system has successfully facilitated the conversion of CG to TA base pairs in plants (Zong et al. 2017), yeast (Nishida et al. 2016), zebrafish (Zhang et al. 2017), and mammalian cells (Billon et al. 2017). Nonetheless, the application of the CBE system in fungi, particularly in filamentous fungi, remains underdeveloped and warrants further exploration. To date, only two implemented instances of the CBE system have been reported in filamentous fungi. The CBE derived from BE3 exhibits an editing efficiency of approximately 60% in Aspergillus niger (Huang et al. 2019a). In contrast, the CBE based on Target-AID demonstrates a gene editing efficiency ranging from 16.67% to 92% in Myceliophthora thermophila (Zhang et al. 2022).

In this study, we report the inaugural development of a mini-SDD7 deaminase-based CBE system in Trichoderma. As we know, this is the first report to demonstrate the feasibility of simultaneous multiplex gene editing by CBE in filamentous fungi. We conducted a comparative analysis of the mini-SDD7 deaminase and the conventional deaminase rAPOBEC1 in T. koningiopsis to evaluate their editing efficiency and site preference for ura5 and yak1. The results demonstrated that rAPOBEC1 achieved an editing efficiency of approximately 13%, whereas mini-SDD7 exhibited an efficiency of 81.3% for yak1, a performance comparable to that observed in mammalian cells (Huang et al. 2023). Consequently, we propose that mini-SDD7 deaminase holds greater potential for further development and application in T. koningiopsis compared to rAPOBEC1. However, during the editing process of yak1 by the mini-SDD7-CBE system, it was observed that an increase in editing efficiency was accompanied by the emergence of C-to-A and C-to-T, as well as partial fragment deletions at the editing sites in certain transgenic lines. These unexpected substitutions likely represent unintended “bystander” mutations occurring within the editor’s activity window at the on-target locus, where multiple nearby cytosines may be deaminated to uracil (U). This uracil is subsequently excised through the combined action of uracil-DNA glycosylase (UNG) and apurinic/apyrimidinic (AP) endonuclease, thereby generating an AP site. During the gap-filling repair process, various DNA damage repair mechanisms are activated, including translesion synthesis (TLS), which may result in unintended base substitutions (Jiang et al. 2021). Furthermore, the repair of multiple damage sites can lead to double-strand breaks, thereby activating the non-homologous end joining (NHEJ) repair pathway, which has the potential to generate indels (Komor et al. 2017). Consequently, there is a need to enhance the expression level of the UNG inhibitor UGI within the mini-SDD7 system. Simultaneously, our findings indicate substantial variability in the editing efficiency of mini-SDD7 across different sites, with the lowest observed efficiency being approximately 10% at the ace1 site and the highest reaching 81.3% at the yak1 site. The primary determinants of base editing efficiency likely include the efficacy of the single-guide RNA (sgRNA) and the accessibility of the target editing sites (Keough et al. 2019). Regarding the base editor, insufficient expression level of UGI may allow UNG to recognize the U:G mismatch. This recognition initiates the base excision repair (BER) pathway by catalyzing the removal of uracil, thereby converting U:G back to C:G (Komor et al. 2017). In our study, the development of a mini-SDD7 deaminase-based CBE in T. koningiopsis holds promise for improving both editing efficiency and precision by optimizing the copy number of UGI and increasing the availability of free UGI molecules. Furthermore, it was reported that the suppression of NHEJ activity could significantly improve editing accuracy and minimize the occurrence of indels (Bae et al. 2020). Beyond on-target byproducts, potential off-target effects should be considered when evaluating overall editing precision. In this study, off-target assessment was limited to a targeted analysis of computationally predicted genomic sites using PCR/Sanger sequencing (25 candidate loci) and therefore did not include unbiased whole-genome sequencing (WGS) or transcriptome-wide RNA-seq analyses. Previous work has shown that Sdd7 cytosine base editors can induce both DNA and RNA off-target effects (Hwang et al. 2025). Accordingly, although we did not detect statistically supported off-target editing at the predicted loci tested under our experimental conditions, we cannot exclude rare or unpredicted genome-wide off-target events, nor RNA off-target editing. Future studies incorporating WGS and RNA-seq will be required to comprehensively define DNA- and RNA-level off-target profiles of mini-SDD7-CBE in filamentous fungi.

The utilization of CBEs in multiplex gene editing offers substantial support in the domains of gene function research (Zuo et al. 2019), crop enhancement (Petherick 2015), and cell therapy (Porreca et al. 2024). However, there are limited applications of multiplex gene editing using base editors in fungi. The mini-SDD7-CBE system achieved an editing efficiency of 83% for yak1, whereas the editing efficiencies for ace1 and pk1 were 10%, respectively. In the context of simultaneous editing of yak1 and ace1, the overall efficiency was 10%. The findings indicate that the multiplex gene editing efficiency of the mini-SDD7-CBE system is likely predominantly determined by the sgRNA exhibiting the lowest editing efficiency. Notably, the concurrent editing of yak1, ace1, and pk1 using the mini-SDD7-CBE system demonstrated that the editing efficiency remained consistent, irrespective of the increased number of target genes, and aligned closely with the efficiency of the least effective sgRNAs. The multiplex gene editing has been successfully accomplished in Yarrowia lipolytica by using the CBE derived from the PmCDA1 deaminase. The efficiency of gene editing markedly declined as the number of targeted genes increased, achieving a maximum efficiency of 83% for single-gene editing and 31% for double-gene editing (Bae et al. 2020). In comparison to PmCDA1, mini-SDD7 demonstrates greater potential for multiplex gene editing. Nonetheless, given the relatively low efficiency of multiplex gene editing observed with the current mini-SDD7-CBE system, there remains significant scope for further enhancement. In addition to enhancing the editing efficiency of sgRNA targeting sites, improving the release efficiency of gRNA is also a critical issue to address in multiplex gene editing (Peng et al. 2020). Recent reports have demonstrated the utilization of multiple endogenous tRNAs, each conjugated with sgRNA targeting distinct genes, within a tRNA-gRNA array to facilitate the creation of a multiplex gene knockout gRNA expression array. This method has successfully enabled CRISPR/Cas9-mediated disruption of one to five target genes in A. niger, achieving efficiencies of 89.2%, 70.91%, 50%, 22.41%, and 4.17%, respectively (Li et al. 2021a). This strategy circumvents the instability associated with the tRNA-gRNA array employed to induce silencing effects and serves as a reference for editing more than three genes using the mini-SDD7-CBE system in T. koningiopsis.

Cellulase production in Trichoderma is subject to regulation by various transcription factors, including transcriptional activators such as Xyr1 (Cao et al. 2019) and Ace2 (Li et al. 2021b), as well as the transcriptional repressors Ace1 (Fang and Chen 2018) and Cre1 (Portnoy et al. 2011). Additionally, their regulation is mediated indirectly through a range of protein kinases, notably serine protein kinase Yak1 (Lv et al. 2015) and MAP kinase Tmk2 (Wang et al. 2014). This study successfully accomplished the concurrent editing of three genes involved in the regulation of cellulose production in T. koningiopsis by the mini-SDD7-CBE system. This approach significantly reduced the time consumption compared to traditional methods involving single genetic manipulations per transformation, thereby facilitating rapid breeding of the industrial strains through genetic modifications. The yield of carboxymethyl cellulase in Δyak1 was 1.6-fold higher than that of WT, while the xylanase yield was increased by 1.3-fold. Similar observations have been documented in T. reesei (Lv et al. 2015). In contrast, deletion of ace1 in T. koningiopsis only led to a modest upregulation of several cellulase genes and did not cause a significant increase in CMCase activity. This suggests that, although Ace1 acts as a transcriptional repressor in many fungi, its impact on overall cellulase production can be constrained by species-specific posttranscriptional or secretory limitations, as well as by the induction conditions (microcrystalline cellulose was used in this study). Inactivation of pk1 resulted in a twofold increase in xylanase yield, but this modification also caused a marked decrease in cellulase production. Consistent with it, cbh1, cbh2, and eg2 were significantly downregulated, whereas the hemicellulase genes xyn1, xyn2, and bxl were strongly induced. Furthermore, the strain Δpk1 demonstrated impaired growth on solid media. This opposite effect on cellulase and hemicellulase production could be reminiscent of the gene-specific regulation of CAZymes by the cell wall integrity (CWI) MAPK pathway as reported in other filamentous fungi, where cellulase genes show little or modest response, while certain xylanase genes (e.g., XYN5 in T. reesei) are markedly activated upon CWIMAPK perturbation (de Paula et al. 2018). Our data therefore suggest that Pk1-dependent signaling may differentially modulate the transcriptional activation and repression of distinct CAZyme gene sets in T. koningiopsis, weakening the expression of major cellulase genes while enhancing that of a subset of hemicellulase genes. These findings imply that the pk1 gene, potentially functioning as a mitogen-activated protein kinase, plays a direct and indirect role in diverse cellular processes, though the precise mechanisms underlying these effects warrant further investigation.

In conclusion, this study represents the first successful construction of a cytosine editing system in Trichoderma. We propose that the mini-SDD7 deaminase is a more suitable component for achieving efficient gene editing compared to the conventional rAPOBEC1 deaminase-based CBE in T. koningiopsis. Utilizing the mini-SDD7-CBE system, we successfully edited one to three genes and obtained strains with significantly enhanced cellulase and xylanase production. Our findings provide a valuable tool for precise genome engineering in Trichoderma species. Such site-specific and multiplexable amino acid editing will facilitate fine-tuning of protein function, stability, and regulation in Trichoderma without introducing double-strand breaks, thereby offering a powerful tool for both functional genomics and industrial strain improvement.

Supplementary Material

Supplementary_Material20260121.docx
TMYC_A_2622835_SM9824.docx (703.6KB, docx)

Funding Statement

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences [XDA28030000] and the National Key Research and Development Program of China [2023YFD1302200].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21501203.2026.2622835

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