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Additional file 7: Figure S6. Verification of quadruple-gene deletions of neo, alp-1, rca-1 and hcr-1 in selected transformants with co-transformation of only four donor-DNAs without CRISPR expressing cassettes. (A) Schematic of homologous recombination (HR) of target genes mediated by each donor DNA. (B) PCR analysis of quadruple-gene deletion of neo, alp-1, rca-1 and hcr-1 in selected transformants using one primer (gh1-1-out-F2, alp1/rca1/hcr1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (gh1-1-in-R2, alp1/rca1/hcr1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of neo, alp1, rca-1 and hcr-1 were 0.8, 1.6, 5.0 and 0.7 kb, respectively, while those of the host strain (rightmost lane) was 1.9, 1.0, 0.6 and 1.0 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout).

Additional file 3: Figure S2. Verification of triple-gene deletions of cre-1, res-1 and gh1-1 in selected transformants by using Pooled single-crRNA-based CRISPR–Cas12a system (A) or crRNA Array-based CRISPR–Cas12a system (B). PCR analysis of triple-gene deletion of cre-1, res-1 and gh1-1 in selected transformants using one primer (cre1/res1/gh1-1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (cre1/res1/gh1-1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of cre-1, res-1 and gh1-1 were 0.8, 0.7 and 1.9 kb, respectively, while those of WT strain (rightmost lane) was 1.2, 0.9 and 1.0 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). The symbol of star indicated deletion mutant. HDR, homology-directed repair; WT, wild type. U6p, U6 promoter; Ptef1, tef1 promoter; TtrpC, trpC Terminator.

Additional file 13: Table S2. List of Cas12a or Cas9 guide and PAM sequences in this study.

Additional file 8: Figure S7. Third round of target genomic editing by CRISPR–Cas12a system. (A) Schematic of homologous recombination (HR) of bar, ap-3 and prk-6 mediated by Cas12a, array2 and donor DNA. (B) PCR analysis of triple-gene deletion of bar, ap3 and prk6 in selected 22 transformants using one primer (alp1-out-F2, ap3/prk6-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (alp1-in-R2, gh1-1/res1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of bar, ap-3 and prk-6 were 0.8, 2.0 and 0.8 kb, respectively, while those of the host strain (rightmost lane) was 2.0, 1.2 and 1.2 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). The symbol of star indicated deletion mutant. HDR, homology-directed repair.

Additional file 12: Figure S11. SDS-PAGE of secreted protein from the eight mutant strains and wild type strain (WT) cultured for 6 days in Avicel inducing medium supplemented with 0.75% yeast extract.

Additional file 6: Figure S5. Second round of target genomic editing by CRISPR–Cas12a system. (A) Schematic of homologous recombination (HR) of neo, alp-1, rca-1 and hcr-1 mediated by Cas12a, array2 and donor DNA. (B) PCR analysis of quadruple-gene deletion of neo, alp-1, rca-1 and hcr-1 in selected transformants using one primer (gh1-1-out-F2, alp1/rca1/hcr1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (gh1-1-in-R2, alp1/rca1/hcr1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of neo, alp1, rca-1 and hcr-1 were 0.8, 1.6, 5.0 and 0.7 kb, respectively, while those of the host strain (rightmost lane) was 1.9, 1.0, 0.6 and 1.0 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). The symbol of star indicated deletion mutant. HDR, homology-directed repair; symbol star indicated deletion mutant.

Additional file 10: Figure S9. Second round of target genomic editing by CRISPR–Cas9 system. (A) Schematic of homologous recombination (HR) of neo, alp-1, rca-1 and hcr-1 mediated by Cas12a, array2 and donor DNA. (B) PCR analysis of quadruple-gene deletion of neo, alp-1, rca-1 and hcr-1 in selected transformants using one primer (gh1-1-out-F2, alp1/rca1/hcr1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (gh1-1-in-R2, alp1/rca1/hcr1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of neo, alp1, rca-1 and hcr-1 were 0.8, 1.6, 5.0 and 0.7 kb, respectively, while those of the host strain (rightmost lane) was 1.9, 1.0, 0.6 and 1.0 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). Symbol star indicated deletion mutant. HDR, homology-directed repair.

Additional file 9: Figure S8. Verification of triple-gene deletions of bar, ap-3 and prk-6 in selected 22 transformants with co-transformation of three donor-DNAs without CRISPR expressing cassettes. (A) Schematic of homologous recombination (HR) of target genes mediated by donor DNA. (B) PCR analysis of triple-gene deletion of bar, ap-3 and prk-6 in selected 22 transformants using one primer (alp1-out-F2, ap3/prk6-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (alp1-in-R2, gh1-1/res1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of bar, ap-3 and prk-6 were 0.8, 2.0 and 0.8 kb, respectively, while those of the host strain (rightmost lane) was 2.0, 1.2 and 1.2 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). HDR, homology-directed repair.

Additional file 1: Table S1. List of PCR primers used in this study.

Additional file 2. PCR analysis of the mutants of M. thermophila generated in this study.

Additional file 1. Primers used for the genetic manipulation in M. thermophila.

Additional file 4: Figure S3. Verification of triple-gene deletions of gh1-1, cre-1 and res-1 in selected transformants with co-transformation of only three donor-DNAs without CRISPR expressing cassettes. (A) Schematic of homologous recombination (HR) of target genes mediated by each donor DNA. (B) PCR analysis of triple-gene deletion of gh1-1, cre-1 and res-1 in selected 23 transformants using one primer (cre1/gh1-1/res1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (cre1/gh1-1/res1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of gh1-1, cre-1 and res-1 were 1.9, 0.8 and 0.7 kb, respectively, while those of the host strain (rightmost lane) was 1.0, 1.2 and 0.9 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout).

Additional file 11: Figure S10. Third round of target genomic editing by CRISPR–Cas9 system. (A) Schematic of homologous recombination (HR) of bar, ap-3 and prk-6 mediated by Cas12a, array2 and donor DNA. (B) PCR analysis of triple-gene deletion of bar, ap-3 and prk-6 in selected 22 transformants using one primer (alp1-out-F2, ap3/prk6-out-F) located upstream of the 5′ flanking region of genomic DNA and the other primer (alp1-in-R2, gh1-1/res1-in-R) located in the 3′ flanking region of genomic DNA. The expected lengths of disrupted transformants of bar, ap3 and prk6 were 0.8, 2.0 and 0.8 kb, respectively, while those of the host strain (rightmost lane) was 2.0, 1.2 and 1.2 kb, respectively. Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). The symbol of star indicated deletion mutant. HDR, homology-directed repair.

Additional file 2: Figure S1. Verification of cre-1 gene deletions in selected transformants with co-transformation of only Cas12a and donor DNA, only crRNA and donor DNA, or only donor DNA. (A) Schematic of homologous recombination (HR) of target gene cre-1. (B–D) PCR analysis of cre-1 deletion with one primer (cre1-out-F) located upstream of the 5′ flanking region of genomic DNA and the other (cre1-in-R) located in the 3′ flanking region of genomic DNA. The expected length of disrupted transformants was 1.9 kb, while that of the WT host strain, used as a negative control, was 1.0 kb (rightmost lane). Heterokaryotic transformants showed two PCR bands (both of wild-type and knockout). HDR, homology-directed repair; WT, wild type.

Additional file 4. Copy number assay by RT-qPCR. cdt-1 in strain JG207cdt; Ctcpp in strain JG412; Mtcpp in strain JG413.

Additional file 15. Nucleotide sequence of the crRNA expression cassettes. Blue letters indicate the RNA polymerase III U6 snRNA promoter. Red letters indicate the 19 nt direct repeat. Green letters indicate the target sequence of amdS, cre-1, res-1, gh1-1, alp-1, neo, rca-1, hcr-1, bar, ap-3, or prk-6, respectively.

Additional file 3. Dry cell weigh of in the culture of the strain strain JG207ΔMtcpp grown on cellobiose and Avicel for 8 days.

Additional file 5: Table S1. Growth rates of wild type and gul-1 mutant. Aliquots of 5 μL 1×107 mL−1 spore suspensions of WT, Δgul-1, Pn-gul-1 and Pc-gul-1 were incubated at 28 °C for 18 h on MM plates.

Additional file 9: Table S4. Primers used in this study.

Additional file 7: Table S3. Comparative analysis of cell wall-related gene expression between Î gul-1 and WT by RNA-seq.

Additional file 10. Nucleotide sequences of donor DNA fragments.

Additional file 8. Nucleotide sequences of Cas9 expression cassettes.

Additional file 1: Figure S1. Phenotype of WT and constitutive Cas9-expressing strains. (A) Sodium dodecylsulfate-polyacrylamide gel electrophoresis of secreted protein of the WT and Cas9-OE after 4 days of culture on 2% Avicel. (B-E) Assays for protein concentration and xylanase, endoglucanase and exoglucanase activities of Cas9-OE and the WT in inducing medium with 2% Avicel after 3 and 4 days culture. (F) Mycelial dry weights of Cas9-OE and the WT from cultures on sucrose and Avicel. No significant difference (Tukey’s HSD, p > 0.5) was observed between Cas9-OE and the WT in the above assays.

Additional file 2: Figure S2. Verification of cre-1 gene deletions in selected transformants with co-transformation of only Cas9 and donor DNA, only sgRNA and donor DNA, or donor DNA alone. (A) Schematic of homologous recombination (HR) of the target gene cre-1. (B-G) PCR analysis of cre-1 deletion in M. thermophila (B-D) and M. heterothallica (E-G) with one primer (cre1-out-F) located upstream of the 5′ flanking region of the genomic DNA and the other (cre1-in-R) located in the 3′ flanking region of the genomic DNA. The expected length of disrupted transformants was 1.9 kb, while that of the host strain, used as a negative control, was 1.0 kb (rightmost lane).

Additional file 7: Table S1. List of PCR primers used in this study.

Additional file 3: Figure S3. Verification of double-gene deletions of cre-1 and res-1, cre-1 and res-1, and gh1-1 and res-1 in selected transformants with co-transformation of multiple fragments. (A) Schematic of homologous recombination (HR) of target genes mediated by Cas9, sgRNAs and donor DNA. (B-D) PCR analysis of double-gene deletion of cre-1 and gh1-1 (B), cre-1 and res-1 (C) and gh1-1 and res-1 (D) in selected transformants using one primer (cre1/gh1-1/res1-out-F) located upstream of the 5′ flanking region of the genomic DNA and the other primer (cre1/gh1-1/res1-in-R) located in the 3′ flanking region of the genomic DNA. The expected length of disrupted transformants was 1.9 kb, while that of the host strain (rightmost lane) was 1.0 kb.

Additional file 9. Nucleotide sequences of sgRNA expression cassettes.

Additional file 6: Figure S6. Determination of PtrpC-neo cassette copy numbers in the disrupted mutants by RT-qPCR analysis.

Additional file 5: Figure S5. Verification of quadruple-locus deletion of cre-1, res-1, gh1-1 and alp-1 in selected transformants. (A) Schematic of homologous recombination (HR) of target genes mediated by Cas9, sgRNA and donor DNA. (B) PCR analysis of quadruple-gene deletion of cre-1, res-1, gh1-1 and alp-1 in selected transformants with paired primers (cre1/res1/gh1-1/alp1-out-F and cre1/res1/gh1-1/alp1-in-R). The expected length of disrupted transformants was 1.9Â kb, while that of the host strain M2 (rightmost lane) was 1.0Â kb.

Additional file 4: Figure S4. Verification of triple-gene deletions of cre-1, res-1, gh1-1 and alp-1 in selected transformants. (A) Schematic of homologous recombination (HR) of cre-1, res-1 and gh1-1 mediated by Cas9, sgRNAs and donor DNA. (B) PCR analysis of triple-gene deletion of cre-1, res-1 and gh1-1 in selected transformants with specific paired primers (cre1/gh1-1/res1-out-F and cre1/gh1-1/res1-in-R). The expected length of disrupted transformants was 1.9Â kb, while that of the host strain (rightmost lane) was 1.0Â kb.

Diagram of tre2_53811 protein from Trichoderma reesei QM6a.

Protein sequence of tre53811 from Trichoderma reesei QM6a.

Phylogenetic tree of alkaline phosphatase proteins.

Primers used in this study.

Primers used in this study.

Protein sequence of the in-house-annotated AP-3 Îź subunit from Trichoderma reesei QM6a.

Phylogenetic tree of alkaline phosphatase proteins.