Multi-gene continuous editing ganoderma lucidum strain based on marker recovery system

Abstract

The invention provides a polygene continuous editing ganoderma lucidum strain Ganoderma lucidum H1-delta 4785 delta 10331 based on a label recovery system, the preservation number is CCTCC M 20252712, and the invention provides a preparation method of the polygene continuous editing ganoderma lucidum strain Ganoderma lucidum H1-delta 4785 delta 10331 and application of the polygene continuous editing ganoderma lucidum strain Ganoderma lucidum H1-delta 4785 delta 10331. Comprising the following steps: step (1), designing and preparing sgRNA, step (2), preparing RNP, step (3), preparing donor DNA, step (4), transforming PEG-mediated ganoderma lucidum protoplast, step (5), screening and verifying a 0Z04785 editing strain, step (6), recovering a screening marker, and step (7), editing the 0Z10331 gene of the 0Z04785 gene editing strain. The H1-delta4785delta10331 strain and the preparation method thereof provided by the invention can be used for researching the influence of knockout of a plurality of isogenes on ganoderma triterpenes or ganoderma growth and the synergistic effect or mutual compensation mechanism of the ganoderma triterpenes or the ganoderma growth in different biological processes, and can also be combined with a breeding technology.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically to a multi-gene sequentially edited Ganoderma lucidum strain based on a label recovery system and its creation method. Background Technology

[0002] Cytochrome P450 (CYP450) is a superfamily of monooxygenases that participate in various metabolic processes in plants and animals, playing a crucial role in growth, development, and stress response regulation. Literature indicates that CYP450 modifies the parent rings of triterpenoids through reactions such as hydroxylation, epoxidation, and carboxylation, significantly enriching the structural diversity of triterpenoids. Due to the large number of members in the P450 gene family, often containing many isoforms, elucidating the function of a target gene typically requires sequential editing of multiple P450 genes to comprehensively and systematically analyze the specific functions of different members within the gene family, as well as their synergistic or compensatory mechanisms in various biological processes.

[0003] Phenotypic analysis is the most direct way to screen transformants to determine whether a gene has been successfully edited. The ura3 gene, encoded by Ganoderma lucidum, is an orotic acid nucleoside-5'-monophosphate decarboxylase (OMP decarboxylase). It is a two-way selection marker; it catalyzes the formation of the highly toxic substance 5-fluorolactalbumin from 5-fluoroorotic acid (5-FOA), thus it can be used for toxicity screening. It also participates in a key reaction in uridine synthesis, so it can be used for auxotrophic screening. When the ura3 gene is deleted, the strain becomes a uridine auxotroph, unable to grow on media without uridine but able to grow on media with 5-FOA. When the ura3 gene is reintroduced, the strain can grow on media without uridine but cannot grow on media with 5-FOA.

[0004] Therefore, it is necessary to research and develop a method for continuous editing of multiple genes in Ganoderma lucidum based on the ura3 marker recovery system and a new strain generated based on this method. Summary of the Invention

[0005] This invention first provides a multi-gene sequentially edited Ganoderma lucidum strain, Ganoderma lucidum H1-Δ4785Δ10331, based on a marker recovery system. This strain was deposited on November 27, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M 20252712, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0006] This invention also provides a method for preparing the above-mentioned multi-gene sequentially edited Ganoderma lucidum strain based on a label recovery system, comprising the following steps:

[0007] Step (1): Design and preparation of sgRNA

[0008] sgRNAs were designed using the CDS regions of the target genes 0Z_04785 and 0Z_10331 (sequences as described in SEQ ID NO.1 and SEQ ID NO.8, respectively), and suitable sgRNAs were screened using an online website (http: / / crispor.tefor.net / ).

[0009] The target sequence is defined as the 20 bp starting 161 bp downstream of the start codon of the 0Z_04785 gene, the target sequence is defined as the 20 bp starting 419 bp downstream of the start codon of the 0Z_04785 gene, the target sequence is defined as the 20 bp starting 174 bp downstream of the start codon of the 0Z_10331 gene, and the target sequence is defined as the 20 bp starting 162 bp downstream of the start codon of the 0Z_10331 gene.

[0010] The target site is located between the third and fourth bases of the target sequence. Using primers (5'→3') sgRNA-F: TTCTAATACGACTCACTATA and sgRNA-R: AAAAGCACCGACTCGGT, sgRNAs containing the T7 promoter are amplified. The sgRNA sequences of target site 1 (0Z_04785), target site 2 (0Z_04785), target site 1 (0Z_10331), and target site 2 (0Z_10331) are as described in SEQ ID NO. 24-27, respectively.

[0011] Step (2): Preparation of RNP

[0012] The RNP complexes were assembled separately to form RNP complexes, which were named RNP-4785-1, RNP-4785-2, RNP-10331-1, and RNP-10331-2, respectively.

[0013] Step (3): Preparation of donor DNA

[0014] The donor DNA sequence for target 1 of gene 0Z_04785 is shown in SEQ ID NO.16, the donor DNA sequence for target 2 of gene 0Z_04785 is shown in SEQ ID NO.17, the donor DNA sequence for target 1 of gene 0Z_10331 is shown in SEQ ID NO.18, and the donor DNA sequence for target 2 of gene 0Z_10331 is shown in SEQ ID NO.19.

[0015] The donor DNA consists of four parts: the homologous upper arm, the marker gene ura3 expression cassette, the loop arm, and the homologous lower arm; the homologous upper arm is the 500 bp sequence upstream of the target site, the homologous lower arm is the 500 bp sequence downstream of the target site, and the loop arm is the 500 bp sequence upstream of the homologous upper arm.

[0016] The homologous upper arm and ura3 were ligated together by overlap PCR and named as follows: 4785-1-UU / 4785-2-UU / 10331-1-UU / 10331-2-UU, which is long fragment 1; the circular arm and homologous lower arm were ligated together and named as follows: 4785-1-DD / 4785-2-DD / 10331-1-DD / 10331-2-DD, which is long fragment 2.

[0017] Step (4): PEG-mediated transformation of Ganoderma lucidum protoplasts

[0018] Protoplasts of strain L1-△ura3 were prepared by activation and liquid culture of strain L1-△ura3 using PDAU and YMGU media, respectively; the protoplasts were then resuspended in STC and diluted to 10⁻⁶. 7 100μL -1 PEG-mediated transformation was performed to obtain single colonies;

[0019] Step (5): Screening and verification of the 0Z_04785 edited strain

[0020] Several single colonies were obtained from screening plates of strain L1-Δura3 for different target sites, and 24 colonies were randomly selected for identification. The fragments amplified in the genome of wild-type strain L1 using primers 4785-1-F / R and 4785-2-F / R were 1957bp and 1961bp in length, respectively, and the fragments amplified in the genome of the transformant were 4857bp and 4858bp in length, respectively.

[0021] According to agarose gel electrophoresis, all three transformants were found to have completely recovered the ura3 selection marker, including one Δ4785-1 strain with target 1 edited, and two Δ4785-2 and Δ4785-3 strains with target 2 edited.

[0022] Step (6): Recycling of filter tags

[0023] Protoplasts were prepared from the positive transformants Δ4785-1, Δ4785-2, and Δ4785-3 with complete ura3 gene expression cassette filling. An appropriate amount of protoplast dilution was evenly spread on YMGAU medium containing 400 mg / L 5-FOA and cultured. Several single colonies were obtained from each. Ten colonies from each were randomly selected and transferred to PDAU medium containing 400 mg / L 5-FOA for secondary screening. Edge hyphae were picked and inoculated onto PDA medium for propagation. Genomic DNA of the transformants was extracted.

[0024] Using primers 4785-1-F / R and 4785-2-F / R, fragments of 1957 bp and 1961 bp were amplified in the genome of the wild-type strain L1, respectively, and fragments of 1337 bp and 1338 bp were amplified in the genome of the transformant, respectively. Based on the agarose gel electrophoresis results, six edited strains were obtained. Four strains were obtained using Δ4785-1 as the test strain: Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, and Δ4785Δura3-4; one strain was obtained using Δ4785-2 as the test strain: Δ4785Δura3-5; and one strain was obtained using Δ4785-3 as the test strain: Δ4785Δura3-6.

[0025] Step (7): Editing of the 0Z_10331 gene in the 0Z_04785 gene-edited strain

[0026] The strains Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, Δ4785Δura3-4, Δ4785Δura3-5, and Δ4785Δura3-6 were activated and cultured in liquid medium using PDAU and YMGU, respectively, to prepare protoplasts of the above six strains. The protoplasts were then resuspended in STC and diluted to 10⁻⁶. 7 100μL -1 PEG-mediated transformation was performed by slowly adding 1 mL of PTC and mixing. The mixture was incubated at 20°C for 30 min, then mixed with 45°C liquid MM medium and poured into plates. The plates were incubated at 26°C for 15-30 days to obtain single colonies. Ten colonies were randomly selected from each colony for identification.

[0027] Using primers 10331-1 / 2-F / R, fragments of 2221 bp were amplified in the genome of the wild-type strain L1, and fragments of 5118 bp were amplified in the genome of the transformants. Agarose gel electrophoresis results showed that the PCR amplification product band size of one of the transformants was consistent with the expectation. Sequencing analysis showed that the transformant completely restored the ura3 selection marker, and it was named H1-Δ4785Δ10331. H1-Δ4785Δ10331 is the desired Ganoderma lucidum strain with continuous multi-gene editing. The strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 27, 2025, with accession number CCTCM20252712, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0028] This invention also provides the application of the above-mentioned multi-gene sequentially edited Ganoderma lucidum strain H1-Δ4785Δ10331 based on the marker recovery system in Ganoderma lucidum breeding. Specifically, it can be used to study the effects of knocking out multiple isoform genes on Ganoderma lucidum triterpenes or Ganoderma lucidum growth, as well as their synergistic effects or mutual compensation mechanisms in different biological processes. It can also be combined with breeding technology to cultivate new Ganoderma lucidum varieties with specific agronomic traits (such as high yield and stress resistance) or novel metabolite profiles.

[0029] Technical effect

[0030] 1. This invention establishes for the first time a CRISPR / Cas9-based ura3 marker recovery system in Ganoderma lucidum and successfully achieves continuous editing of two cyp450 genes, 0Z_04785 and 0Z_10331.

[0031] 2. In L1 mononuclear cells, the content of ganoderic acid did not change after the 0Z_04785 gene was knocked out, but the content of ganoderic acid S decreased when both the 0Z_04785 and 0Z_10331 genes were knocked out at the same time, suggesting that the 0Z_10331 gene is related to the synthesis of ganoderic acid S.

[0032] 3. By sequentially editing multiple cyp450 genes, we can not only fully reveal the specific functions and interaction mechanisms of each member in this family, but also help identify functional redundancy or specificity in gene families.

[0033] Many traits of Ganoderma lucidum are controlled by multiple genes with minor effects, making the systematic study of the functions of these genes of great significance. This invention, for the first time, utilizes a marker-recovery system in Ganoderma lucidum to achieve the sequential knockout of multiple genes, which is of great value for elucidating the genetic basis of traits such as triterpenoid synthesis in Ganoderma lucidum. The multi-gene sequentially edited Ganoderma lucidum strain provided by this invention is based on a ura3-based uridine auxotrophic marker-recovery system. By designing an expression cassette containing the ura3 gene and two repetitive sequences, under 5-FOA selection pressure, the marker gene ura3 was precisely looped out of the host genome. This system provides feasibility for the recycling of selection markers in Ganoderma lucidum and lays a theoretical foundation for the research of multi-gene editing technology.

[0034] Furthermore, homology comparison revealed that the 0Z_04785 and 0Z_10331 genes are highly similar to the genes cyp512a3 and cyp512v2, which are co-expressed with the key triterpenoid synthesis gene lss, respectively. Studies have also shown that the content of ganoderic acid T in silencing strains of cyp512v2 is significantly reduced. This invention targets the 0Z_04785 and 0Z_10331 genes and investigates their relationship with the biosynthesis of Ganoderma lucidum triterpenoids through sequential editing of these two P450 genes. Attached Figure Description

[0035] Figure 1 A gel map was constructed by labeling recyclable homologous arm carriers.

[0036] Note: M: D5000 DNAMarker.

[0037] Figure (a): 1-2 are the homologous upper arms, 3-4 are the ura3 expression boxes, 5-6 are the loop arms, and 7-8 are the homologous lower arms;

[0038] Figure (b): 1-5 are long segment 1, and 6-10 are long segment 2.

[0039] Figure 2 Screening of the 0Z_04785 gene-edited strain.

[0040] Note: Figure (a): MM screening plate of RNP-4785-1;

[0041] Figure (b): MM screening plate of RNP-4785-2.

[0042] Figure 3 PCR results of colony-derived transformants of the 0Z_04785 gene after editing.

[0043] Note: The gray box indicates that the strip size is consistent with expectations.

[0044] M: D5000 DNA Marker; S1: Primer 4785-1-F / R amplifies the genome of wild-type strain L1; S2: Primer 4785-2-F / R amplifies the genome of wild-type strain L1; 7: strain Δ4785-1; 16: strain Δ4785-2; 21: strain Δ4785-3.

[0045] Figure 4 Screening for positive transformant marker loopout.

[0046] Note: Figure (a): Strain Δ4785-1; Figure (b): Strain Δ4785-2; Figure (c): Strain Δ4785-3.

[0047] Figure 5 PCR detection results of colonies after positive transformant marker looping.

[0048] Note: M: 5000 molecular weight standard.

[0049] Figure (a): 1 is wild-type strain L1, 2 is strain Δ4785-1 marked before looping, and 3-6 are strains Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3 and Δ4785Δura3-4 marked after looping;

[0050] Figure (b): 1 is wild-type strain L1, 2-3 are strains Δ4785-2 and Δ4785-3 before the labeling loop, and 4-5 are strains Δ4785Δura3-5 and Δ4785Δura3-6 after the labeling loop.

[0051] Figure 6 Phenotypic characteristics of the ura3 selection marker before and after looping in the 0Z_04785 gene-edited strain.

[0052] Note: CK represents L1 monokaryotic strain, and WT represents L1-ura3 monokaryotic strain. Figure (a): Before the marker loops out; Figure (b): After the marker loops out.

[0053] Figure 7 Screening of the 0Z_10331 gene-edited strain.

[0054] Note: Strains Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, Δ4785Δura3-4, Δ4785Δura3-5, and Δ4785Δura3-6 correspond to screening plates for different target sites in 0Z_10331.

[0055] Figure 8 PCR detection results of the 0Z_10331 gene-edited strain.

[0056] Note: M: 5000 molecular weight standard; 1: strain L1; 2: strain H1-Δ4785Δ10331.

[0057] Figure 9 Phenotypic characteristics of the ura3 selection marker loop before the 0Z_10331 gene-edited strain.

[0058] Note: CK represents L1 monokaryotic strain, and WT represents L1-ura3 monokaryotic strain.

[0059] The transformant H1-Δ4785Δ10331 before tagging could not grow on PDA medium containing 5-FOA, but could grow on MM medium, and its phenotype was consistent with that of wild-type strain L1.

[0060] Figure 10 HPLC results of the 0Z_04785 single-gene edited strain and the 0Z_04785 and 0Z_10331 double-gene edited strains.

[0061] Note: Figure (a): 1-4 are HPLC chromatograms of the edited strain at ganoderic acid Mk, S, T and R, respectively;

[0062] Figure (b): (i)-(iv) are peak area difference analysis diagrams of the edited strain at ganoderic acid Mk, S, T and R, respectively.

[0063] The results showed a significant difference between the double gene-edited strain and the wild-type strain (**P<0.01). Detailed Implementation

[0064] The following embodiments are merely further explanations and illustrations of the technical solutions protected by this invention, and are not intended to limit the scope of protection of this invention.

[0065] Sources of materials and reagents

[0066] 1.1 Test strains

[0067] The binuclear strain of G. lucidum 'Hunong No.1' G0119 and the monouclear strain L1 isolated from G0119 were provided by the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences.

[0068] The ura3 gene of mononuclear strain L1 was edited into strain L1-Δura3. The preparation method is described in the literature: Tian Jialin, Dong Beibei, Tang Chuanhong, et al. Method for creating homozygous mutant strains of Ganoderma lucidum gene loci using CRISPR / Cas9 gene editing and hybridization technology [J]. Journal of Edible Fungi, 2025, 32(03):1-12. DOI:10.16488 / j.cnki.1005-9873.2025.03.001.

[0069] Escherichia coli DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0070] 1.2 Main Reagent Sources

[0071] Glucose, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and mannitol (Shanghai Sinopharm Chemical Reagent Co., Ltd.); PDA powder (BD Biosciences, USA); LB powder (Haibo Biotechnology Co., Ltd.); yeast extract and malt extract (Oxoid, UK); vitamin B1, asparagine, uridine, and 5-fluoroorotic acid (5-FOA) (Shanghai Sangon Biotech Co., Ltd.); sorbitol and low-melting-point agarose (Shanghai Shaoxin Biotechnology Co., Ltd.); calcium chloride and polyethylene glycol (PEG) 4000 (Sigma-Aldrich, USA); Tris-HCl (Beijing Regen Biotechnology Co., Ltd.); Triton X-100 (BioFroxx, Germany); lysozyme (Institute of Microbiology, Guangdong Academy of Sciences); HiScribe T7 Quick High Yield RNA Synthesis Kit RNA (New England Biolabs, USA); RNAClean & Concentrator-25 (Zymo, USA). Research Company); NLS-Cas9 Nuclease, 10×Reaction Buffer (Suzhou Nearshore Protein Technology Co., Ltd.); DNA Extraction Kit, Fungal Total RNA Mini-Prep Kit (Guangzhou Meiji Biotechnology Co., Ltd.); 2XEasy Taq8PCR SuperMix (Beijing TransGen Biotechnology Co., Ltd.); 6×Loading Buffer (Guangzhou Dongsheng Biotechnology Co., Ltd.); Ultra One Step Cloning Kit (C115) Seamless Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.)

[0072] 1.3 Preparation of culture medium and other solutions

[0073] Ampicillin stock solution (L) -1 ): 100g ampicillin powder.

[0074] Urea yeast maltose medium (YMGU) (L) -1 ): 10g malt extract, 4g yeast extract, 4g glucose, 100mg uridine.

[0075] Urea yeast maltose selection medium (YMGAU) (L) -1 ): 10g malt extract, 4g yeast extract, 4g glucose, 100mg uridine, 10g low melting point agarose.

[0076] Potato Culture Medium (PDA) (L) -1 ): 39g PDA powder.

[0077] Urate potato broth (PDAU) (L) -1 ): 39g PDA powder, 100mg uridine.

[0078] Basic culture medium (MM)(L) -1 ): 109.3g mannitol, 20g asparagine, 20g glucose, 10g low-melting-point agar, 1.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.46g potassium dihydrogen phosphate, 0.125mg vitamin B1.

[0079] Urate basal medium (MMU) (L) -1 ): 109.3g mannitol, 20g asparagine, 20g glucose, 10g low-melting-point agar, 1.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 0.46g potassium dihydrogen phosphate, 0.125mg vitamin B1, 100mg uridine.

[0080] Sorbitol Tris(hydroxymethyl)aminomethane Calcium Chloride Buffer (STC)(L) -1 ): 0.55 mol sorbitol, 10 mmol calcium chloride, 10 mmol Tris-HCl (pH 7.5).

[0081] Polyethylene glycol tris(hydroxymethyl)aminomethane calcium chloride buffer (PTC) (L) -1 ): 600g PEG4000, 10mmol Tris-HCl (pH 7.5) and 50mmol calcium chloride.

[0082] SEQ ID NO.1 gene 0Z_04785 fragment (5'→3') 2119bp:

[0083] ATGGCAGTGGAGGATCCTCAAGCTCTCATTCTCGCCGGCATCGCCATCCTCGCTGTCCTA

[0084] TACGTCGTCCGATGGCAGACAGACCCCGTAAGTGGGTCCCTCACAAAAGCGCTATGCGGT

[0085] CAATGATAAACTAATTATCCGAACAGCTGAGGTCTATCCCCACAGTTGGTGGGCCGTCC

[0086] GCACCAGGACTATCGATGCTGTCAGCGCTCAATTACCTGCGTAATGGGAAGAAGGTGAT

[0087] GACTGAGGGCTACCGGAAGGTCTGTCTATCTTCTCCACCGGCAGCTCCGCAGGGACTC

[0088] TGCAGGAGCTCACAAATCCTGTTTAATAGTACCACGGAGGAGCCTTCAAGGTCGCACA

[0089] GCTCGACAAATGGGTCGTCGTGGTTTCCGGGCGCAAGTTGGTCGACGAACTCTGGCGG

[0090] CGACCGGACGAGGATTTATCGGCTCCTGCGGCTGTTCAGGACGTACGAATACGCATGCA

[0091] GCCGCACATCTTGTCCTACATTGACTTGGTCTCCTTCGTAGATCATCCAGATGAGATATA

[0092] CTCTCGGACACGAAGCATTTGATGACCCGTATGAAACCGACATCATCAAGGAGAAGCTT

[0093] ACGCGTTCGCTTCCGGTCATTTTTCCTGACGTCGTCGAGGAGATGAGGCTTGCCGTGTC

[0094] GGACTACATCCCCACAAAAGGAGACGGTAAGCAGTCTTTCAACCAGACCCTCTTTTGA

[0095] CGGGCTCTCAATTGAATTCACAAAGAGTGGACCCCTGTCAATGGTATGGACACGGCGG

[0096] TGAATATTGTCGCAAGAACCAGTAGCCGCGCCTTCGTGGGTCTCCCACTTTGTAGGTCC

[0097] TCGTTTTCACTTCATCAGCATACCGACCAGCTCATGCGGAAACGCTTCAGGCCGCAACG

[0098] AAGAATTCTTGGCGTTGATTCTTCGTTTCACTATGGATGTGTTGAAGGATCGGTTCTTTC

[0099] TCAACACTTTCCCGTACTTTTTGAAGCCGTGAGTCCAGTCTTGGAAAGTAATGACAGGA

[0100] TTTCTCACGTTCTCTGGACAGTTACTTCGGGCACGCCTTCAGTAGAGCGAAGAGGACTA

[0101] TCTATCAAGGACTGACATTCCCCAACCGTTGATCACCGAAAGAAGGATGAATATGCGA

[0102] GAGCTTGGGGACGACTGGTCTGGACAAGCCGGTAAGTGTCCCGTCGAGGGTCTGCGAC

[0103] AATGTGACTAAAGAGTGCCTGTTGCAGGTTGATATGCTTCAGTGGGTCGTGGAAGCGG

[0104] CGATGGCCAGGAAGAGCGATGATTATAGGATTGCGGAACGCATGTTCCTCGTTAACTTC

[0105] GCAGCTCTCCACACACCTCCACCGTGCGCCCAATCTTCCCTGTCCTGCCAAGCGGTAC

[0106] TAATACCCTAGTTCAACCCGTATAGACCCTTGCGCACGCGCTCTATGATCTCGCTGCCAT

[0107] GCCCGAGCTCATTCCAGAACTGCGGGAAGAGGTCGATTCCGCCATCGCGTCGGACGGT

[0108] TGGTCCAAAGCGGCTGTAGGCAAAATGTGGAAGCTCGATAGCGTGTGCAGAGAGGCGT

[0109] TGCGTTACCATGGAATGAGCTTCAGTGTGTATCATATTATCTATTACACGAGCAGGCTCA

[0110] GCGGTTCCCTGACTGACGGCCTACACCCCCCCCCTCCCACCAGTCGGTCTTTTCCGCAA

[0111] GGCAATGAAGGACGTCACGCTGAGCGACGGGACATTCATCCCGAAAGGCACCACCGTA

[0112] GTCGCCGCAGCAGGCCCCACGCACCACGAGGCGTCGATCTACCCCAACCCGGAGGTG

[0113] CTCGACCCCTTCCGCTTCGCAAAGTTGGGTGCAAGTGGCGGAGAAGGCGGTTCCGTGA

[0114] AGCTCCAAACCGTTTCCACGTCCATTGATTTCCTGCCCTTCGGCCATGGCAAGCACGCT

[0115] TGGTGTGTCCCCTCTCCGCCACGCCCTGCTCGTGCCAAAGCAAGCGTTCTGACTGCCAT

[0116] ACAGCCCGGGACGGTGGTTCGCGGCAAACGAGGTGAAGATGATCTTAGCGCACATCGT

[0117] GCTCAACTACGACCTGAAGCTCGGTGGAGACGGCAAACGGCCTGGGGACACGCTCTT

[0118] CGGTACCACCATACTTCCGCCCCGTGGGCAGGTGTATTTCAGGAAACGCAAAGGGGTG

[0119] CCCGAGTGA

[0120] SEQ ID NO.2 Gene 0Z_04785 Target 1 Homologous Upstream Arm Fragment 500bp (5'→3'): TATGTCCCCCGTTCCTCATTCTCGCTCGATGGCATGATCAACCTCGCTTCCCAGGCTTTACATCGGGGCATGCACGGTCATCCAGGGGCATGCGTGGCAACTTAAATCGACATGGGCTTCCAGCAACCCGGCGAAAACAGACATTCCTGTGCGGTCTGAGCAAATGAAGCAAGTGCCCTACCAAACCATCCTATATCTCGGAGGCGCGGTCACCTGGATCAGTAGATGTGAGTAAGGACCATTACACACGCAACCTTCCATACGCCCCAGAGTTGTCCGAGTTGGCAGCAGGCATGCGTCGCCATAAGTATCCTCTATCACCTTCCGCCTGTCTTGTTACTTCTCTCCCCTCTCAACCAGCTGGACCGTTGGACTCGTGCCCCATCTCTCTGATGGCAGTGGAGGATCCTCAAGCTCTCATTCTCGCCGGCATCGCCATCCTCGCTGTCCTATACGTCGTCCGATGGCAGACAGACCCCGTAAGTGGGTCCTCACAAAGC

[0121] SEQ ID NO.3 Gene 0Z_04785 Target 1 Homologous Lower Arm Fragment 500bp (5'→3'): AAGGTGATGACTGAGGGCTACCGGAAGGTCTGTCTATCTTCTCCACCGGCAGCTCCGCAGGGACTCTGCAGGAGCTCACAAATCCTGTTTAATAGTACCACGGAGGAGCCTTCAAGGTCGCACAGCTCGACAAATGGGTCGTCGTGGTTTCCGGGCGCAAGTTGGTCGACGAACTCTGGCGGCGACCGGACGAGGATTTATCGGCTCCTGCGGCTGTTCAGGACGTACGAATACGCATGCAGCCGCACATCTTGTCCTACATTGACTTGGTCTCCTTCGTAGATCATCCAGATGAGATATACTCTCGGACACGAAGCATTTGATGACCCGTATGAAACCGACATCATCAAGGAGAAGCTTACGCGTTCGCTTCCGGTCATTTTTCCTGACGTCGTCGAGGAGATGAGGCTTGCCGTGTCGGACTACATCCCCACAAAAGGAGACGGTAAGCAGTCTTTCAACCAGACCCTCTTTTGACGGGCTCTCAATTGAATTCACAA

[0122] SEQ ID NO.4 Gene 0Z_04785 Target 1 Loop-out Arm 500bp (5'→3'):

[0123] GAGAATGTGGGGTGCGGGGAGTGAGAACGCGGTAGTCGGAGGGAGAGGGCCGACCG

[0124] GTGACGACGATGTCGGACAGATTACGGTTAGGTGGCATGGCGCATCGATATCGAGCGCA

[0125] AACCAAGACGAAGAGGAGGCGAAATGCGAGGAGGGCAGCGTTGCCAGCCGTTGTCCG

[0126] GTGTCGCGCGGGAAGGAGTGTGTATTCTGAGCTATTTTCGCTGTAAAGGTTAAACAGCT

[0127] GCGACGGCAAGGCCGCTGGTGGCCAGAGCGTGGAACTTGAAGAGCGTGGACCGCGCG

[0128] TGGACTGCGGTGTGGAGGCGAGCGAGATCTGGCAGAGGAGATTGTGCTGCACAGACC

[0129] GGTTGTTATGGTCACCGCCAACTGAGCCAGATCCCCGAGGCGATCGAGCCGCCTGCGG

[0130] GATCTTCCGCTACGGCGCTGCTCTCCTTGCTTCCTTGTCTTATGTAGTACGAGTGTCTTA

[0131] CGCCTCTTCATCCCCATTTATATTGCACCATGGAATT

[0132] SEQ ID NO.5 Gene 0Z_04785 Target 2 Homologous Upper Arm Fragment 500bp (5'→3'): TTCCATACGCCCCAGAGTTGTCCGAGTATTTATGTCCCCCGTTCCTCATTCTCGCTCGATGGCATGATCAACCTCGCTTCCCAGGCTTTACATCGGGGCATGCACGGTCATCCAGGGGCATGCGTGGCAACTTAAATCGACATGGGCTTCCAGCAACCCGGCGAAAACAGACATTCCTGTGCGGTCTGAGCAAATGAAGCAAGTGCCCTACCAAACCATCCTATATCTCGGAGGCGCGGTCACCTGGATCAGTAGATGTGAGTAAGGACCATTACACACGCAACCTTCCATACGCCCCAGAGTTGTCCGAGTTGGCAGCAGGCATGCGTCGCCATAAGTATCCTCTATCACCTTCCGCCTGTCTTGTTACTTCTCTCCCCTCTCAACCAGCTGGACCGTTGGACTCGTGCCCCATCTCTCTGATGGCAGTGGAGGATCCTCAAGCTCTCATTCTCGCCGGCATCGCCATCCTCGCTGTCCTATACGTCGTCCGATGGCAG

[0133] SEQ ID NO.6 Gene 0Z_04785 Target 2 Homologous Lower Arm Fragment 500bp (5'→3'): ACATTGACTTGGTCTCCTTCGTAGATCATCCAGATGAGATATACTCTCGGACACGAAGCATTTGATGACCCGTATGAAACCGACATCATCAAGGAGAAGCTTACGCGTTCGCTTCCGGTCATTTTTCCTGACGTCGTCGAGGAGATGAGGCTTGCCGTGTCGGACTACATCCCCACAAAAGGAGACGGTAAGCAGTCTTTCAACCAGACCCTCTTTTGACGGGCTCTCAATTGAATTCACAAAGAGTGGACCCCTGTCAATGGTATGGACACGGCGGTGAATATTGTCGCAAGAACCAGTAGCCGCGCCTTCGTGGGTCTCCCACTTTGTAGGTCCTCGTTTTCACTTCATCAGCATACCGACCAGCTCATGCGGAAACGCTTCAGGCCGCAACGAAGAATTCTTGGCGTTGATTCTTCGTTTCACTATGGATGTGTTGAAGGATCGGTTCTTTCTCAACACTTTCCCGTACTTTTTGAAGCCGTGAGTCCAGTCTTGGA

[0134] SEQ ID NO.7 Gene 0Z_04785 Target 2 Loop-out Arm 500bp (5'→3'):

[0135] AGAGCGTGGAACTTGAAGAGCGTGGACCGCGCGTGGACTGCGGTGTGGAGGCGAGCG

[0136] AGATCTGGCAGAGGAGATTGTGCTGCACAGACCGGTTGTTATGGTCACCGCCAACTGA

[0137] GCCAGATCCCCGAGGCGATCGAGCCGCCTGCGGGATCTTCCGCTACGGCGCTGCTCTCC

[0138] TTGCTTCCTTGTCTTATGTAGTACGAGTGTCTTACGCCTCTTCATCCCCATTTATATTGCA

[0139] CCATGGAATTTATGTCCCCCGTTCCTCATTCTCGCTCGATGGCATGATCAACCTCGCTTC

[0140] CCAGGCTTTACATCGGGGCATGCACGGTCATCCAGGGGCATGCGTGGCAACTTAAATCG

[0141] ACATGGGCTTCCAGCAACCCGGCGAAAACAGACATTCCTGTGCGGTCTGAGCAAATGA

[0142] AGCAAGTGCCCTACCAAACCATCCTATATCTCGGAGGCGCGGTCACCTGGATCAGTAGA

[0143] TGTGAGTAAGGACCATTACACACGCAACC

[0144] SEQ ID NO.8 Gene 0Z_10331 Fragment (5'→3') 2198bp:

[0145] ATGGCGGACGACCAAGCCGTCGGGTTCATCTGCATCGGTATTCTGGTTGGCATCGTCGT

[0146] ATACCGGTGGAACATCAGCCCCGTAAGTGGTTCCCCCGCATATGGTGAAGGCGACCTAC

[0147] TGCTGAGTGGGACCACTCATAGTTGGACGACATACCGACCGTGGGGGGTTCCTGGGTG

[0148] ACAGGCCTGTCGTATCTCCCAGCATTTCTCTGGACCCGGCGGTTGAGGGAGCTGTTTTT

[0149] CGAAGGTTACAAGAAGGTACGCATGCTCGTTCCCCCGTTCGCTTGTGTGAAAAGGTCT

[0150] GAACCTAAACGGCTATCGGTGACAGTACTACGGCTCTGCGTTCAAGATCCCTTTGCTGG

[0151] ACCGGTGGCTTGTCATCGTGTCAGGCCCGAAGATGATTGAGGACATCAGACGACGACC

[0152] TGAAGACGAGCTGTCCTTCACCGAGGCCATCCAAACAGTCAGTCCTTGCTTATGATCTA

[0153] CGTGACGATCCTGGCGTCGTTATTACGGGGCTTACCACATTTCTAGCTTCTGCAATACAG

[0154] ATACAACGTCGGCTGGAAGATGCGAGATGATCCGTACCACATCACCATCGTGAAGGAG

[0155] AGATTGCAGAATAAGATGCTGCCAGCTATCATGCCCGACTTGATAGATGAGGTGGGCCA

[0156] TGCGGTACAAAGGTCTATTCCAGAAAGGGGGAATGGTGGGTCATCTTATTGCTTATACT

[0157] GGGGTAACGGCTTGGTTTGATCACAGATATCTCTAGAATGGGTCGCTATGGACGTGTCG

[0158] TCGGCCGCGTTGAGGGTCGTTGCGCGGACGTGCAACCGCGCCTTTGTGGGACTACCTC

[0159] TCTGTATGTTGTTCTGCCCTCTCTGAATTCGAAGGACTTTTTATTGATTGGCTCTTAAATT

[0160] CAGGTCGGAACGAAGAGTACAACGACCTCTGTGCGAAATTCACTGTCGAAATTCTCAA

[0161] AACTGCAAGAGCTCTGCGAATCTTCCCTGATTTGCTTAAATCGTTAGTACTAACTCAATG

[0162] AACCGCTCCGTCTATCCGGCATCGAACTTACTCTTTGTCGTGCCCATGCAGGATTGCAG

[0163] CCCGTTTCATGTGCAAAACAGTAAAGCAGACGAAGGACCGCGCCCTCTTGCGCCTCCG

[0164] GCCGGTCATCCGTGGGCGGATAGCGGCGACCCTACGAGAGAAGGACAAGGAACGGTT

[0165] CTACAGACAGGTATGTGTCAGGTAATTCTAAACGGGTTCTGCAGTGGGTATTGAGCGGC

[0166] CTTGTTCATGTAGAATGACGTCCTGCAATCGATCGTTGATAGAGCAGTGATCAGGCACG

[0167] AGACAGACATCGACATTACAGAAAGGCTGCTGCTGCTTAACCTCGCCGCCATCCACAC

[0168] GTCCTCCAACGTGCGTGCTCCTGGGTTCTTTCCTTTTAAAAAAAAATGACACTCTGCCC

[0169] TTTTTTCAGAGCTTGACTCAAGTGCTGTACCACCTTGCGGAACGCCCCGAGCTTCTCGT

[0170] GCCTCTCAGAGAGGAGATCGAAGCGGCCATAGACGTAGAGGGCTGGACGATGCACTCC

[0171] TTCGCCCGCATGTGGAAGCTCGACAGCCTCCTCCGCGAGTCCGAGCGGTACAACGGGT

[0172] TCACCATCGGTACGCCCGTTCTGTCCTTCCCTTTTCTTCTCTCTCCCAAGTTTGAACGAG

[0173] CGAAGGCGACTAACGCGTGGCCGATTTTCAGCATCCCTCATGCGCCTCGCGCGGAAGG

[0174] ACGTCATCCTCGAAAACGGCATGCTCATCCCGAAGGGCACCATCCTCGGCGCGCCCGC

[0175] GCACCCGATGCACCACGACGACGCGCACTTCCCGAACGCGGACGTCTTTGACCCGTTC

[0176] CGCTTCGCGCGCATGCGCGAGGCCGCGTGCGACTCGGAGGCGTCCGCACGCCACCAGT

[0177] TCGCGAGCACCTCGCCCGGATACATCGCGTTTGGCCACGGGCAGCTTGCTTGGTGAGT

[0178] CCGCAAGGCGTCGAGCACTGGCCAGGTGAACGTTAACATGGAACTAAAAATGTGAATG

[0179] TCAACGTAGTCCAGGGAGATTCTTCGCCGCCAACCAGCTCAAGGCGGTGCTCTCGTAC

[0180] ATCATCCTCAACTATGATCTGAAGCTCCGCGAGCCGGACTCAGATGGCGACAACCCTCG

[0181] GCGCCCTCCAAACGAGTACGTCTCGATCGCGGTTCTCCCTCCAGCGGGTGGGACCATC

[0182] CTCGTGAGGAAGCGCGCAACCCTCGCCTAA

[0183] SEQ ID NO.9 Gene 0Z_10331 Target 1 Homologous Upper Arm Fragment 500bp (5'→3'): TTACAAGAAGGTACGCATGCTCGTTCCCCCGTTCGCTTGTGTGAAAAGGTCTGAACCTAAACGGCTATCGGTGACAGTACTACGGCTCTGCGTTCAAGATCCCTTTGCTGGACCGGTGGCTTGTCATCGTGTCAGGCCCGAAGATGATTGAGGACATCAGACGACGACCTGAAGACGAGCTGTCCTTCACCGAGGCCATCCAAACAGTCAGTCCTTGCTTATGATCTACGTGACGATCCTGGCGTCGTTATTACGGGGCTTACCACATTTCTAGCTTCTGCAATACAGATACAACGTCGGCTGGAAGATGCGAGATGATCCGTACCACATCACCATCGTGAAGGAGAGATTGCAGAATAAGATGCTGCCAGCTATCATGCCCGACTTGATAGATGAGGTGGGCCATGCGGTACAAAGGTCTATTCCAGAAAGGGGGAATGGTGGGTCATCTTATTGCTTATACTGGGGTAACGGCTTGGTTTGATCACAGATATCTCTAG

[0184] SEQ ID NO.10 Gene 0Z_10331 Target 1 Homologous Lower Arm Fragment 500bp (5'→3'): CTAGAGATATCTGTGATCAAACCAAGCCGTTACCCCAGTATAAGCAATAAGATGACCCACCATTCCCCCTTTCTGGAATAGACCTTTGTACCGCATGGCCCACCTCATCTATCAAGTCGGGCATGATAGCTGGCAGCATCTTATTCTGCAATCTCTCCTTCACGATGGTGATGTGGTACGGATCATCTCGCATCTTCCAGCCGACGTTGTATCTGTATTGCAGAAGCTAGAAATGTGGTAAGCCCCGTAATAACGACGCCAGGATCGTCACGTAGATCATAAGCAAGGACTGACTGTTTGGATGGCCTCGGTGAAGGACAGCTCGTCTTCAGGTCGTCGTCTGATGTCCTCAATCATCTTCGGGCCTGACACGATGACAAGCCACCGGTCCAGCAAAGGGATCTTGAACGCAGAGCCGTAGTACTGTCACCGATAGCCGTTTAGGTTCAGACCTTTTCACACAAGCGAACGGGGGAACGAGCATGCGTACCTTCTTGTAA

[0185] SEQ ID NO.11 Gene 0Z_10331 Target 1 Loop-out Arm 500bp (5'→3'):

[0186] TCGACCGTACATTCATGCATACGGCCAGCGCTTGATTTGTGGAAGCAAGGTGGCGCTCA

[0187] GAGTAGGTAACGCCAGGCTAGTGTGGGGTAGGATATAGGTAAGTATCCAGGTTCGACTA

[0188] TCTCAACATTGCTAATGGGAACCTCGCGTCTATCCGACGTCATTAATCCCGCTTCCAAGG

[0189] CCGCGAATCGTTCATTCCCAAGGCTACATGCGTTCCAGCATTGACTCCGCCTGCAACAC

[0190] CGTGCATGATCATGCTTCATTTGTGCCCAACGCGCTCCTGGTATACGGGCTTACGAATAT

[0191] AGATCATCTATCATGTCACATCAAGTCGTGAGCGGGACGCGGACTTCCAAACATGTACG

[0192] CCCTCCCCTTATCTCTTGGCGGGTCGCCTGCGTATTCGCCTCTCACGACGGAAGGCGAG

[0193] CATACAACCGATGAGCGGACCTACAATTCGTCATGTCCCTCCCTTTGACTCCCCGGTTC

[0194] GAGGATCCTAGTTCTACAACTGGACG

[0195] SEQ ID NO.12 Gene 0Z_10331 Target 2 Homologous Upper Arm Fragment 500bp (5'→3'): TTCTACAACTGGACGCCCATTCAAGCATTACGCACGCACGACGGCATCCATCGGATCCCGAACATTCTTCCTGCCGCCATGTGGCTAGCGCCCTTCTCGTAACGTTTCCTTCGCACTGAGACCGGCAGGCTTGCACTTTCCCAACTGAGGGGTAGTCGCTGTTGTGACCCTGAGGCAACAAGCGCAATCGGCTCTCGATTTTTACTGCAGGTGGGTTCCATGCGCCTTACCTCCACCACCTGGTACTGACTCCAAAGGCTTGCTCGGACGTCATACCTTATTCAGGACCGCGCACGTACATTTTCGAGACCTATGATGCGATCGCCGCTCGCTCGTCCTTTTAAAAGGCTCACAGACGTTATCGTCCCTTTTGCCAGCACCTTCCGATCCAACGATGGCGGACGACCAAGCCGTCGGGTTCATCTGCATCGGTATTCTGGTTGGCATCGTCGTATACCGGTGGAACATCAGCCCCGTAAGTGGTTCCCCCGCATATGGTG

[0196] SEQ ID NO.13 Gene 0Z_10331 Target 2 Homologous Lower Arm Fragment 500bp (5'→3'): GTTTTTCGAAGGTTACAAGAAGGTACGCATGCTCGTTCCCCCGTTCGCTTGTGTGAAAAGGTCTGAACCTAAACGGCTATCGGTGACAGTACTACGGCTCTGCGTTCAAGATCCCTTTGCTGGACCGGTGGCTTGTCATCGTGTCAGGCCCGAAGATGATTGAGGACATCAGACGACGACCTGAAGACGAGCTGTCCTTCACCGAGGCCATCCAAACAGTCAGTCCTTGCTTATGATCTACGTGACGATCCTGGCGTCGTTATTACGGGGCTTACCACATTTCTAGCTTCTGCAATACAGATACAACGTCGGCTGGAAGATGCGAGATGATCCGTACCACATCACCATCGTGAAGGAGAGATTGCAGAATAAGATGCTGCCAGCTATCATGCCCGACTTGATAGATGAGGTGGGCCATGCGGTACAAAGGTCTATTCCAGAAAGGGGGAATGGTGGGTCATCTTATTGCTTATACTGGGGTAACGGCTTGGTTTGATCAC

[0197] SEQ ID NO.14 Gene 0Z_10331 Target 2 Loop-out Arm 500bp (5'→3'):

[0198] TTGTCCTAGGGCCCGTCGACCGTACATTCATGCATACGGCCAGCGCTTGATTTGTGGAA

[0199] GCAAGGTGGCGCTCAGAGTAGGTAACGCCAGGCTAGTGTGGGGTAGGATATAGGTAAG

[0200] TATCCAGGTTCGACTATCTCAACATTGCTAATGGGAACCTCGCGTCTATCCGACGTCATT

[0201] AATCCCGCTTCCAAGGCCGCGAATCGTTCATTCCCAAGGCTACATGCGTTCCAG CATTG

[0202] ACTCCGCCTGCAACACCGTGCATGATCATGCTTCATTTGTGCCCAACGCGCTCCTGGTAT

[0203] ACGGGCTTACGAATATAGATCATCTATCATGTCACATCAAGTCGTGAGCGGGACGCGGA

[0204] CTTCCAAACATGTACGCCCTCCCCTTATCTCTTGGCGGGTCGCCTGCGTATTCGCCTCTC

[0205] ACGACGGAAGGCGAGCATACAACCGATGAGCGGACCTACAATTCGTCATGTCCCTCCC

[0206] TTTGACTCCCCGGTTCGAGGATCCTAG

[0207] SEQ ID NO.15 gene ura3 sequence 2520bp (5'→3'):

[0208] CTCCACTCCCTCCAACTATTTTCGCCTGTCGGCCTGCCCAGCCGCTGTCACTCACGAAT <�

[0209] GGCGGTTCTGGTCCCTCAAGATACAGGTCCTTGACACGGGCAATAAGGGCTTCTGACA

[0210] AGAAGGGTTTGAGTGATTCAGTGAAGCGTTCGGGCCATGGCTCGTCCTCTTGTGGAGT

[0211] GTCCCCGTCAACTTTTCGTTCATCCGTTGCGGGTGGCTGCGCGGCGTCCGGTGCGGGGT

[0212] CGGCGACAACAGCGTCGCTAACTTGCTCTGCAGTGGTGGCAGAAGTTTCGGGCTCGTT

[0213] GTTCTCACCGACGGCTTGATCAGCTGGCTTAGACGTAGTGGAGACTCCTGCAGGCGTC

[0214] It should be noted that in the original text, there is a misspelling in the tag "<�

[0209] ", which is likely a typo and should probably be "

[0209] ". This has been maintained in the translation as it is part of the original text.ACATCCGCCCCGTTCTTCGATGAGGATGGCATAGCGAGCGTCTTCAAATGGATGACCTG

[0215] ACTGTCTTGGTCGACCTCGAATACCAAGAAGTCCGTGAACCTGGAAGGCCATATGAAC

[0216] TAAATAAACGAACGCGAGTATGACCTTACCACGCACCTCTGCTTGATGATGCCCTCGAT

[0217] TTTGGGCACATCAAAACCAATGTACTCAGATATACCAACGTCCGTTTCCATGATTTGCTG

[0218] TATAGAGCCATCTGGACCATGCACTGGGGGTTTTGTGTTGAGCAGCGCATGACTTGGCG

[0219] GAAGAAGAGTCTCCGGTTCGTCGTTCTTCACGTCAACGTCCATATCGTCCTGTTCCTGC

[0220] TCTTCGACGTCGGCGATGCTGACAACCGTGACGGTGCTGTCCGTGCTCATAGGCATGAC

[0221] TCCGTCCCCGTCCTCTACTTTTGCTCGCTTGGCATTCGGTTCATCCGCTAGATCTGGCTC

[0222] GCGAGGGCGAGTTTTTGGCGAGGACGACATTCGGTGTCAGTGAAGCGGAGGGGAGTA

[0223] GAGACGGTCACTGGAGAGATGGGAGAGCCTCGTTCGAGGCAAAACTGGAAAACAGCG

[0224] CGCTTATCTAATCTCACAAGTCACCCAGCAACCACCCGTCGGCCACAAAACAAAATTG

[0225] AAAGAATGGTGGCCGTGGCCAAGCAAACATACGCGCAGAGGGCCACCAGACATCCCA

[0226] ACCCAGCTGCGAAAGCTCTCCTCGAGACGATCGAGCGCAAGCGCACAAATCTGTCTGT

[0227] TAGCGTCGATGTGACGAAACGGGAGGACTTCTTCAGGATTGTGGACATCGTGGGTCCAT

[0228] ACGTCTGCCTAGTAAAGGTAGGTGTACTATGTCCAAATGGTTGATATGCACTTGGGTGA

[0229] ATATAATCCAAAGACCCACATAGACATCATCGAGGACTTTGATCCATCCGCGATCGAAC

[0230] GCCTCAAGGCACTCAGCGAGAAGCATGACTTCCTCATCTTTGAAGACAGAAAATTTGC

[0231] CGACATTGGTAGGATGTAGTCCCTTTGCGCCCGCTACATACGATGTTGACAAGAATATAG

[0232] GAAACACGGTGGCACTTCAATATTCTGCAGGCGTGCACAAAATCGCTAGCTGGTCGCA

[0233] CATCACGAACGCACACCCTGTCCCAGGACCGTCCATCATCTCCGGCCTCAAGGCTGTC

[0234] GGTCTGCCCCTTGGCAGGGGGCTTCTGCTCCTCGCCGAGATGAGCACCAAGGGCAGCC

[0235] TCGCAACCGGCTCATACACGGAAGAGGCCGTCCGTATGGCTCGCGCCAACCGCGACTT

[0236] CGTGATCGGCTTCATTGCCCAGCAACGCATGGACAGCGTCGGTCTGCGGGAGGGCGAG

[0237] TCCTCGCCGGACGAAGACTTCCTTATCCTTACTCCAGGAGTCGGACTGGATACTAGGGG

[0238] AGATAGCATGGGGCAGCAATACCGAACGCCAAGGGAGGTGGTCATCGAGTCCAATTGC

[0239] GATGTCATCATAGTTGGTCGGGGTGTTTACGGGAACGATAACGGCACGAATGCGGAGG

[0240] CAGTCCGCGCACAGGCGGAGAGGTACCGCGCAGAAGGCTGGAAGGCATACCAGGAAA

[0241] GGGTTGGGATCTCGGATTAGGGCTAGCATTGTGCAGAGACGGGTATAGTAGCACAGTGT

[0242] ATATGTAAACAAAACATCCTACCGCTACAATCGCAATACATATACACATACTTATCGTTAA

[0243] ATCAGGCAGTCGTTCCTTCCACTTCCGCCTTCCTTCTCTCCTCTTCCTCTTTGGCTTCGC

[0244] GCTCTTCTTCCTCTACAACTTCGATGTCCAGTGGGAAGGTCAAAATTGCAGCGATGCCC

[0245] GTTAGTTGGTTCAACTCTGCATGAAATTGACCATGTCAGTGGTACGGTAAGCATCAGGA

[0246] TGGGAAAACTCTTACGCTGGCCAGACTCGTGCATACTAGAGAAGATCAAGACCTCCCC

[0247] GCCTTTCTGTTGGACGTCTTCCACTATGCGCACGTACTTTTTGCGCACCGTAGCGTTGCT

[0248] TGACCTGGGATTATGATCAAGACTACGTTCATCGGAAGGAGTCAGCTGGGCTAACCTGA

[0249] ACAACTCGTCCGAGATCATAAGTGTGCCGATGGCGCCTCGATCTGAAGCAAGAGCGAC

[0250] ATGATCGGGACCATACCAGGCTCGCATCTCGTCCGAGGCTAGCATCTTGAAGAACCTGTG

[0251] SEQ ID NO.16 Donor DNA sequence of gene 0Z_04785 target 1, 4020 bp (5'→3'):

[0252]

[0253]

[0254]

[0255]

[0256] Unless otherwise specified, all gene fragment sequences described in this article are in the 5'→3' direction.

[0257] Example 1: Establishment and application of a Ganoderma lucidum multi-gene sequentially edited strain based on a label recovery system

[0258] Step (1): Design and preparation of sgRNA

[0259] sgRNAs were designed using the CDS regions of the target genes 0Z_04785 and 0Z_10331 (sequences as described in SEQ ID NO.1 and SEQ ID NO.8, respectively), and suitable sgRNAs were screened using an online website (http: / / crispor.tefor.net / ). The design of sgRNAs should avoid homology with non-target sequences to ensure target specificity.

[0260] The target sequence 1 is 20 bp starting 161 bp downstream of the start codon of the 0Z_04785 gene: CACAGTTGGTGGGCCGTCCG (SEQ ID NO.20); the target sequence 2 is 20 bp starting 419 bp downstream of the start codon of the 0Z_04785 gene: GACGAGGATTTATCGGCTCC (SEQ ID NO.21); the target sequence 1 is 20 bp starting 174 bp downstream of the start codon of the 0Z_10331 gene: GGGTGACAGGCCTGTCGTAT (SEQ ID NO.22); and the target sequence 2 is 20 bp starting 162 bp downstream of the start codon of the 0Z_10331 gene: TGGGGGGTTCCTGGGTGACA (SEQ ID NO.23).

[0261] The target site (the cleavage site of the Cas9 protein) is located between the third and fourth bases of the target sequence. Using primers (5'→3') sgRNA-F: TTCTAATACGACTCACTATA and sgRNA-R: AAAAGCACCGACTCGGT, sgRNAs containing the T7 promoter were amplified, with sequences as described in SEQ ID NO. 24-27. In vitro transcription and purification of the sgRNA were performed according to the instructions of the HiScribe T7 Quick High Yield RNA Synthesis Kit and the RNAClean & Concentrator kit.

[0262] The sgRNA sequence for target 1 of 0Z_04785 is as follows:

[0263] TCTAATACGACTCACTATA CACAGTTGGTGGGCCGTCCG GTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG CTTTT (SEQ ID NO.24, synthesized by Suzhou Genewise Biotechnology Co., Ltd.).

[0264] The sgRNA sequence of target 2 of 0Z_04785 is as follows:

[0265] TCTAATACGACTCACTATA GACGAGGATTTATCGGCTCC GTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTT (SEQ ID NO.25, synthesized by Suzhou Genewise Biotechnology Co., Ltd.).

[0266] The sgRNA sequence of target 1 at 0Z_10331 is as follows:

[0267] TCTAATACGACTCACTATA GGGTGACAGGCCTGTCGTAT GTTTTAGAGCTAGAAATA GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC TTTT (SEQ ID NO.26, synthesized by Suzhou Genewise Biotechnology Co., Ltd.).

[0268] The sgRNA sequence of target 2 of 0Z_10331 is as follows:

[0269] TCTAATACGACTCACTATA TGGGGGGTTCCTGGGTGACA GTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG CTTTT (SEQ ID NO.27, synthesized by Suzhou Genewise Biotechnology Co., Ltd.).

[0270] Step (2): Preparation of RNP

[0271] The RNP complexes were assembled on ice in a 20 μL reaction system: 6 μg NLS-Cas9 Nuclease (Suzhou Nearshore Protein Technology Co., Ltd.), 3.6 μg sgRNA prepared in step (1) and 2 μL 10× Reaction buffer were mixed and made up to 20 μL with nuclease-free water. The mixture was then incubated at 37℃ for 15 min to form RNP complexes (named RNP-4785-1, RNP-4785-2, RNP-10331-1 and RNP-10331-2, respectively).

[0272] Step (3): Preparation of donor DNA

[0273] The donor DNA used in the multi-gene sequential editing based on the label recovery system includes the donor DNA sequence for target 1 of gene 0Z_04785 as shown in SEQ ID NO.16, the donor DNA sequence for target 2 of gene 0Z_04785 as shown in SEQ ID NO.17, the donor DNA sequence for target 1 of gene 0Z_10331 as shown in SEQ ID NO.18, and the donor DNA sequence for target 2 of gene 0Z_10331 as shown in SEQ ID NO.19. The ura3 sequence is shown in SEQ ID NO.15.

[0274] Each donor DNA consists of four parts: a homologous upper arm, a marker gene ura3 expression cassette, a loop arm, and a homologous lower arm. The homologous upper arm is the 500 bp upstream of the target site, the homologous lower arm is the 500 bp downstream of the target site, and the loop arm is the 500 bp upstream of the homologous upper arm.

[0275] The homologous upper arm of gene target 1 of 0Z_04785 was amplified using primers 4785-1-UP-F and 4785-1-UP-R; the ura3 sequence of gene target 1 of 0Z_04785 was amplified using primers 4785-1-URA3-F and 4785-1-URA3-R; the loop arm sequence of gene target 1 of 0Z_04785 was amplified using primers 4785-1-DR-F and 4785-1-DR-R; and the homologous lower arm sequence of gene target 1 of 0Z_04785 was amplified using primers 4785-1-DOWN-F and 4785-1-DOWN-R. Figure 1 (as shown in (a)) The sequences of the homologous upper arm, homologous lower arm, and loop arm of target 1 of 0Z_04785 are shown in SEQ ID NO.2-4, respectively.

[0276] The homologous upper arm of target 2 of the 0Z_04785 gene was amplified using primers 4785-2-UP-F and 4785-2-UP-R; the ura3 sequence of target 2 of the 0Z_04785 gene was amplified using primers 4785-2-URA3-F and 4785-2-URA3-R; the loop arm sequence of target 2 of the 0Z_04785 gene was amplified using primers 4785-2-DR-F and 4785-2-DR-R; and the homologous lower arm sequence of target 2 of the 0Z_04785 gene was amplified using primers 4785-2-DOWN-F and 4785-2-DOWN-R. (Amplification diagram and...) Figure 1 (a) The sequences of the homologous upper arm, homologous lower arm, and loop arm of target 2 of 0Z_04785 are shown in SEQ ID NO.5-7, respectively.

[0277] The homologous upper arm of target site 1 of the 0Z_10331 gene was amplified using primers 10331-1-UP-F and 10331-1-UP-R; the ura3 sequence of target site 1 of the 0Z_10331 gene was amplified using primers 10331-1-URA3-F and 10331-1-URA3-R; the loop arm sequence of target site 1 of the 0Z_10331 gene was amplified using primers 10331-1-DR-F and 10331-1-DR-R; and the homologous lower arm sequence of target site 1 of the 0Z_10331 gene was amplified using primers 10331-1-DOWN-F and 10331-1-DOWN-R. (Amplification diagram and...) Figure 1 (a) The sequences of the homologous upper arm, homologous lower arm, and loop arm of target 1 at 0Z_10331 are shown in SEQ ID NO. 9-11, respectively.

[0278] The homologous upper arm of target site 2 of the 0Z_10331 gene was amplified using primers 10331-2-UP-F and 10331-2-UP-R; the ura3 sequence of target site 2 of the 0Z_10331 gene was amplified using primers 10331-2-URA3-F and 10331-2-URA3-R; the loop arm sequence of target site 2 of the 0Z_10331 gene was amplified using primers 10331-2-DR-F and 10331-2-DR-R; and the homologous lower arm sequence of target site 2 of the 0Z_10331 gene was amplified using primers 10331-2-DOWN-F and 10331-2-DOWN-R. (Amplification diagram and...) Figure 1 (a) The sequences of the homologous upper arm, homologous lower arm, and loop arm of target 2 at 0Z_10331 are shown in SEQ ID NO. 12-14, respectively.

[0279] The PCR amplification system for the ura3 fragment is as follows: template (L1 genome) 1 μl, primer 4785-1-URA3-F (or 4785-2-URA3-F / 10331-1-URA3-F / 10331-2-URA3-F) 1 μl, primer 4785-1-URA3-R (or 4785-2-URA3-R / 10331-1-URA3-R / 10331-2-URA3-R) 1 μl, 2×Easy PCR SuperMix 25 μl, ddH2O 22 μl. The PCR amplification program for the ura3 fragment was: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 2 min 30 sec, and 72℃ final extension for 5 min.

[0280] The PCR amplification system for the homologous upper and lower arms was as follows: template (L1 genome) 1 μl, primers 4785-1-UP-F (4785-1-DOWN-F / 4785-2-UP-F / 4785-2-DOWN-F / 10331-1-UP-F)

[0281] -F / 10331-1-DOWN-F / 10331-2-UP-F / 10331-2-DOWN-F)1μl, primer 4785-1-UP-R(4785-1-DOWN-R / 4785-2-UP-R / 4785-2-DOWN-R / 10331-1-UP

[0282] -R / 10331-1-DOWN-R / 10331-2-UP-R / 10331-2-DOWN-R) 1μl, 2×Easy PCR SuperMix 25 μl, ddH2O 22 μl. The PCR amplification program for the homologous upper and lower arms was as follows: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 30 sec, and 72℃ final extension for 5 min.

[0283] The homologous upper arm and ura3 were ligated together using overlap PCR and named as follows: 4785-1-UU / 4785-2-UU / 10331-1-UU / 10331-2-UU (long fragment 1); the circular arm and homologous lower arm were ligated together and named as follows: 4785-1-DD / 4785-2-DD / 10331-1-DD / 10331-2-DD (long fragment 2). Figure 1 (b)).

[0284] The overlap PCR amplification system for 4785-1-UU / 4785-2-UU / 10331-1-UU / 10331-2-UU is as follows: template (1 μl each of homologous upper arm and ura3), 1 μl of the upstream primer of the homologous upper arm, 1 μl of the downstream primer of ura3, and 2×Easy... PCR SuperMix 25 μl, ddH2O 21 μl. Overlap PCR amplification program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 3 min, and 72℃ final extension for 5 min.

[0285] The overlap PCR amplification system for 4785-1-DD / 4785-2-DD / 10331-1-DD / 10331-2-DD is as follows: template (1 μl each for the looped arm and the homologous lower arm), 1 μl of the upstream primer for the looped arm, 1 μl of the downstream primer for the homologous lower arm, and 2×Easy primers. PCR SuperMix 25 μl, ddH2O 21 μl. Overlap PCR amplification program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 1 min, and 72℃ final extension for 5 min.

[0286] The homologous recombination system consisted of: linearized vector pCE-Zero 79.14 ng, 4785-1-UU / 4785-2-UU / 10331-1-UU / 10331-2-UU 60.4 ng, 4785-1-DD / 4785-2-DD / 10331-1-DD / 10331-2-D-D 20 ng, 2×ClonExpress Mix 5 μl, and ddH2O to bring the total volume to 10 μl. The system was heated at 50 °C for 5 min and immediately cooled on ice to obtain the recombinant product. Thaw DH5α chemically competent cells on ice. Add 10 μl of recombinant product to 100 μl of competent cells, gently tap the tube wall to mix, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 sec, and immediately cool on ice for 2-3 min. Add 900 μl of LB liquid medium, shake at 37°C for 1 h (200 rpm), centrifuge at 500 rpm for 5 min, discard 900 μl of supernatant, resuspend the bacterial cells in the remaining medium, and gently spread evenly on LB solid medium plates containing ampicillin using a sterile spreader. Incubate upside down in a 37°C incubator for 12-16 h, transform into E. coli, and select single clones for sequencing.

[0287] Source of the vector pCE-Zero: Ultra One Step Cloning Kit (C115) Seamless Cloning Reagent Kit (Nanjing Novizan Company)

[0288] Homologous upper arm primers for target 1 of 0Z_04785:

[0289] Primer 4785-1-UP-F: TTCGGATTCCAGAGATATCTATGTCCCCCGTTCCTCATTCTC

[0290] Primer 4785-1-UP-R:

[0291] GGCTAGCATCTTGAAGAACCTGGTGGCTTTGTGAGGACCACTTACG

[0292] ura3 primer for target 1 of 0Z_04785:

[0293] 4785-1-URA3-F:CGTAAGTGGGTCCTCACAAAGCCACAGGTTCTTCAAGATGCTAGCC

[0294] Primer 4785-1-URA3-R: CCCGCACCCCACATTCTCCTCCACTCCCTCCAACTATTTTCG0Z_04785 Loop arm primer for target 1:

[0295] Primer 4785-1-DR-F: CGAAAATAGTTGGAGGGAGTGGAGGAGAATGTGGGGTGCGGG Primer 4785-1-DR-R: GGTAGCCCTCAGTCATCACCTTAATTCCATGGTGCAATATAAATGGGGATG

[0296] Homologous lower arm primers for target 1 of 0Z_04785:

[0297] Primer 4785-1-DOWN-F: CATCCCCATTTATATTGCACCATGGAATTAAGGTGATGACTGAGGGCTACC

[0298] Primer 4785-1-DOWN-R: CAACTGCCGTCGACGATATCTTGTGAATTCAATTGAGAGCCCGTCAAA

[0299] Homologous upper arm primers for target 2 of 0Z_04785:

[0300] Primer 4785-2-UP-F: TTCGGATTCCAGAGATATCTTCCATACGCCCCAGAGTTGTC

[0301] Primer 4785-2-UP-R: GGCTAGCATCTTGAAGAACCTGTGATTTGTCGAGCTGTGCGACCTTG

[0302] ura3 primer for target 2 of 0Z_04785:

[0303] 4785-2-URA3-F:CAAGGTCGCACAGCTCGACAAATCACAGGTTCTTCAAGATGCTAGCC

[0304] Primer 4785-2-URA3-R: ACGCTCTTCAAGTTCCACGCTTCTCCACTCCCTCCAACTATTTTCG

[0305] Loop arm primer for target 2 of 0Z_04785:

[0306] Primer 4785-2-DR-F: CGAAAATAGTTGGAGGGAGTGGAGAGCGTGGAACTTGAAGAGCGT

[0307] Primer 4785-2-DR-R:GATCTACGAAGGAGACCAAGTCAATGTGGTTGCGTGTGTAATGGTCCTTAC

[0308] Homologous lower arm primers for target 2 of 0Z_04785:

[0309] Primer 4785-2-DOWN-F: GTAAGGACCATTACACACGCAACCACATTGACTTGGTCTCCTTCGTAGATC

[0310] Primer 4785-2-DOWN-R: CAACTGCCGTTCGACGATATCTCCAAGACTGGACTCACGGCTT

[0311] Homologous upper arm primers for target 1 of 0Z_10331:

[0312] Primer 10331-1-UP-F:TTCGGATCTTCCAGAGATATCCCCATTCAAGCATTACGCACGC

[0313] Primer 10331-1-UP-R: AGGCTAGCATCTTGAAGAACCTGTGGCAGTAGGTCGCCTTCACCAT

[0314] ura3 primers for gene target 1 of 0Z_10331:

[0315] Primer 10331-1-URA3-F:ATGGTGAAGGCGACCTACTGCCACAGGTTCTTCAAGATGCTAGCCT

[0316] Primer 10331-1-URA3-R: CCGTATGCATGAATGTACGGTCGACTCCACTCCCTCCAACTATTTTCG;

[0317] Loop arm primer for 0Z_10331 gene target 1:

[0318] Using primer 10331-1-DR-F: CGAAAATAGTTGGAGGGAGTGGAGTCGACCGTACATTCATGCATACGG

[0319] Primer 10331-1-DR-R: GAACGAGCATGCGTACCTTCTTGTAACGTCCAGTTGTAGAACTAGGATCC

[0320] Homologous lower arm primers for gene target 1 of 0Z_10331:

[0321] Primer 10331-1-DOWN-F:

[0322] GGATCCTAGTTCTACAACTGGACGTTACAAGAAGGTACGCATGCTCGTTC

[0323] Primer 10331-1-DOWN-R:

[0324] CAACTGCCGTTCGACGATATCCTAGAGATATCTGTGATCAAACCAAGCC.

[0325] Homologous upper arm primers for target 2 of 0Z_10331:

[0326] Primer 10331-2-UP-F:

[0327] TTCGGATCTTCCAGAGATATCTTCTACAACTGGACGCCCATTCAAGC

[0328] Primer 10331-2-UP-R:

[0329] AGGCTAGCATCTTGAAGAACCTGTGCACCATATGCGGGGGAACCACTT

[0330] ura3 primers for gene target 2 of 0Z_10331:

[0331] Primer 10331-2-URA3-F:

[0332] AAGTGGTTCCCCCGCATATGGTGCACAGGTTCTTCAAGATGCTAGCCT

[0333] Primer 10331-2-URA3-R:

[0334] TCGACGGGCCCTAGGACAACTCCACTCCCTCCAACTATTTTCG;

[0335] Loop arm primer for 0Z_10331 gene target 2:

[0336] Primer 10331-2-DR-F:

[0337] CGAAAATAGTTGGAGGGAGTGGAGGTTGTCCTAGGGCCCGTCGA

[0338] Primer 10331-2-DR-R:

[0339] GCGTACCTTCTTGTAACCTTCGAAAAACCTAGGATCCTCGAACCGGGGA

[0340] Homologous lower arm primers for gene target 2 of 0Z_10331:

[0341] Primer 10331-2-DOWN-F:

[0342] TCCCCGGTTCGAGGATCCTAGGTTTTTCGAAGGTTACAAGAAGGTACGC

[0343] Primer 10331-2-DOWN-R:

[0344] AACTGCCGTTCGACGATATCGTGATCAAACCAAGCCGTTACCC.

[0345] Step (4): PEG-mediated transformation of Ganoderma lucidum protoplasts

[0346] Protoplasts of strain L1-△ura3 were prepared by activation and liquid culture of strain L1-△ura3 using PDAU and YMGU media, respectively. Mycelia were collected by filtration, washed with sterile water and 0.6 mol / L mannitol, and then incubated with 2% (w:v) lysozyme at 30°C for 3 h. Protoplasts were collected by centrifugation at 1258 g for 6 min. The protoplasts were resuspended in STC and diluted to 10⁻¹⁰. 7 100μL -1 PEG-mediated transformation was performed: 100 μL of STC (containing 10...) was added... 7 Protoplasts), 20 μL RNP-4785-1 / 2 (prepared in step (2)), 10 μL donor DNA (donor DNA of 0Z_04785 target 1 / 2, prepared in step (3)), 50 μL PTC and Triton X-100 to a final concentration of 0.006% were mixed and incubated on ice for 10 min. 1 mL PTC was slowly added and mixed, and incubated at 20 °C for 30 min. After mixing with 45 °C liquid MM medium, the mixture was poured into plates and cultured at 26 °C for 15-30 days to obtain single colonies. Figure 2 ).

[0347] Step (5): Screening and verification of the 0Z_04785 edited strain

[0348] Several single colonies of strain L1-Δura3 were obtained from screening plates corresponding to different target sites, and 24 colonies were randomly selected for identification. Using primers 4785-1-F / R and 4785-2-F / R, the amplified fragments in the wild-type strain L1 genome were 1957 bp and 1961 bp in length, respectively, and the amplified fragments in the transformant genome were 4857 bp and 4858 bp in length, respectively. The PCR amplification system was as follows: template (transformant genomic DNA) 1 μl, upstream primer 4785-1-F / 4785-2-F 1 μl, downstream primer 4785-1-R / 4785-2-R 1 μl, 2×Easy PCR SuperMix 25 μl, ddH2O 22 μl. PCR amplification program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 59℃ annealing for 30 sec, 72℃ extension for 5 min, and 72℃ final extension for 5 min.

[0349] Agarose gel electrophoresis results showed that the PCR amplification products of three of the transformants had band sizes consistent with expectations. Figure 3 Sequencing analysis showed that all three transformants had fully recovered the ura3 selection marker, including one strain with target 1 edited (Δ4785-1) and two strains with target 2 edited (Δ4785-2 and Δ4785-3).

[0350] Primer 4785-1-F: TGCTGAGTCCTCCAGTCGTT,

[0351] 4785-1-R:ACTGGACTCACGGCTCAAA;

[0352] 4785-2-F:TGTTTGTAGCTCGACTTCGG、

[0353] 4785-2-R: ACTGTCCAGAGAACGTGAGA.

[0354] Step (6): Recycling of filter tags

[0355] Protoplasts were prepared from the positive transformants Δ4785-1, Δ4785-2, and Δ4785-3, which had their ura3 gene expression cassette fully restored. Appropriate amounts of protoplast dilution were evenly spread onto YMGAU medium containing 400 mg / L 5-FOA and incubated at 26°C for 14 days. Several single colonies were obtained from the selection plates of strains Δ4785-1, Δ4785-2, and Δ4785-3. Figure 4 Ten transformants were randomly selected from each culture and transferred to PDAU medium containing 400 mg / L 5-FOA for secondary screening. They were cultured at 26°C for 7 days. Edge hyphae were picked and inoculated onto PDA medium for propagation, and genomic DNA of the transformants was extracted.

[0356] Fragments amplified using primers 4785-1-F / R and 4785-2-F / R in the genome of wild-type strain L1 were 1957 bp and 1961 bp in length, respectively, and fragments amplified in the genome of the transformant were 1337 bp and 1338 bp in length, respectively. The PCR amplification system was as follows: template (transformant genomic DNA) 1 μl, upstream primers 4785-1-F / 4785-2-F 1 μl, downstream primers 4785-1-R / 4785-2-R 1 μl, 2×Easy PCR SuperMix 25 μl, ddH2O 22 μl. PCR amplification program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 59℃ annealing for 30 sec, 72℃ extension for 5 min, and final extension at 72℃ for 5 min. Agarose gel electrophoresis results showed that the PCR amplification products of 6 transformants had band sizes consistent with expectations. Figure 5 ).

[0357] Sequencing analysis showed that the ura3 selection marker was successfully looped out in all 6 transformants. Four strains (Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3 and Δ4785Δura3-4) were obtained using Δ4785-1 as the test strain, one strain (Δ4785Δura3-5) was obtained using Δ4785-2 as the test strain, and one strain (Δ4785Δura3-6) was obtained using Δ4785-3 as the test strain.

[0358] Transformants that were not tagged with a loop (Δ4785-1, Δ4785-2, and Δ4785-3) could not grow on PDA medium containing 5-FOA, but could grow on MM medium, exhibiting a phenotype consistent with wild-type strain L1. Figure 6 (a)). Conversely, the transformed strains after labeling and looping (Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, Δ4785Δura3-4, Δ4785Δura3-5, and Δ4785Δura3-6) were able to grow on PDA medium containing 5-FOA but not on MM medium, and their phenotype was consistent with that of the test strain L1-Δura3. Figure 6 (b) The results showed that the strain before the labeling loop was able to synthesize uridine on its own and restore the function of the ura3 gene; while the strain after the labeling loop could not express the ura3 gene normally and showed uridine nutritional deficiency. Therefore, it can be used as an experimental strain for subsequent gene function studies.

[0359] Step (7): Editing of the 0Z_10331 gene in the 0Z_04785 gene-edited strain

[0360] Protoplasts of strains Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, Δ4785Δura3-4, Δ4785Δura3-5, and Δ4785Δura3-6 were prepared by activation and liquid culture using PDAU and YMGU media, respectively. Mycelia were collected by filtration, washed with sterile water and 0.6 mol / L mannitol, and then 2% (w:v) lysozyme was added. The mixture was incubated at 30°C for 3 h, and the protoplasts were collected by centrifugation at 1258 g for 6 min. The protoplasts were resuspended in STC and diluted to 10⁻⁶. 7 100μL -1 PEG-mediated transformation was performed: 100 μL of STC (containing 10...) was added... 7Protoplasts), 20 μL LRNP-10331-1 / 2 (obtained in step (2)) and the corresponding 10 μL donor DNA (donor DNA of 0Z_10331 target 1 / 2, obtained in step (3)), 50 μL LPTC and Triton X-100 to a final concentration of 0.006% were mixed and incubated on ice for 10 min. 1 mL LPTC was slowly added and mixed, and incubated at 20℃ for 30 min. After mixing with 45℃ liquid MM medium, the mixture was poured into plates and cultured at 26℃ for 15-30 days to obtain single colonies. Figure 7 Ten samples were randomly selected from each group for identification.

[0361] The PCR amplification fragments were 2221 bp in the wild-type strain L1 genome and 5118 bp in the transformant genome using primers 10331-1 / 2-F / R. The PCR amplification system consisted of: 1 μl template (transformant genomic DNA), upstream primer 10331-1 / 2-F, downstream primer 10331-1 / 2-R, and 2× Easy primers. PCR SuperMix 25 μl, ddH2O 22 μl. PCR amplification program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 sec, 59℃ annealing for 30 sec, 72℃ extension for 5 min, and final extension at 72℃ for 5 min. Agarose gel electrophoresis results showed that the PCR amplification product band size of one of the transformants was consistent with expectations. Figure 8 Sequencing analysis showed that the transformant fully recovered the ura3 selection marker, and it was named H1-Δ4785Δ10331. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 27, 2025, with accession number CCTCM 20252712. The deposit address is No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, 430072, China.

[0362] The transformant H1-Δ4785Δ10331, before tagging and looping, could not grow on PDA medium containing 5-FOA, but could grow on MM medium, exhibiting a phenotype consistent with the wild-type strain L1. Figure 9 The results showed that strain H1-Δ4785Δ10331 was able to synthesize uridine on its own, restoring the function of the ura3 gene.

[0363] 10331-1 / 2-F:GAAGCACGAGATCAGTAGCC、

[0364] 10331-1 / 2-R: TGTACTCTTCGTTCCGACCT.

[0365] Example 2: Effects of the 0Z_04785 and 0Z_10331 genes on the synthesis of Ganoderma lucidum triterpenes

[0366] The obtained single-gene-edited strain 0Z_04785 (Δ4785-1), the double-gene-edited strain 0Z_04785 and 0Z_10331 (H1-Δ4785Δ10331), and the wild-type strain L1 were fermented separately, and Ganoderma lucidum triterpenes were extracted for HPLC analysis. The specific method is as follows: The three revived Ganoderma lucidum strains were inoculated into primary fermentation liquid medium. The cultured liquid inoculum was poured into a sterile homogenizing cup and homogenized twice at low speed for 7 seconds. 10 mL of the homogenate was inoculated into secondary fermentation liquid medium and cultured at 26℃ and 150 rpm for 5 days with constant temperature shaking. The culture was then allowed to stand at 26℃ for 14 days. The upper mycelium was collected and freeze-dried. The freeze-dried mycelium was extracted with anhydrous ethanol at a material-to-liquid ratio of 1:20 (w / v), sonicated for 1.5 h, centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm organic filter membrane for sample loading. The ethanol extract was loaded for liquid chromatography analysis at a flow rate of 1.0 mL / min, an injection volume of 10 μL, a column temperature of 30℃, and a detection wavelength of 254 nm. The HPLC elution program is shown in Table 1. The results showed that different strains exhibited four distinct characteristic peaks within the retention time range of 35–60 min. Figure 10 (a) Based on the peak positions of the ganoderic acid standard, peaks 1-4 were identified as ganoderic acid Mk, ganoderic acid S, ganoderic acid T, and ganoderic acid R, respectively. Figure 10 (b), corresponding to (i), (ii), (iii), and (iv) respectively.

[0367] Compared with the wild-type strain L1, the peak areas of the four ganoderic acids in strain Δ4785-1 did not change significantly, while the peak area of ​​ganoderic acid S in strain H1-Δ4785Δ10331 decreased significantly by 51.78%. Figure 10 The results showed that the 0Z_04785 gene was not significantly correlated with the synthesis of the four ganoderic acids, while the 0Z_10331 gene was significantly correlated with the synthesis of ganoderic acid S.

[0368] Table 1. HPLC elution procedure for triterpenes in alcohol extract.

[0369]

Claims

1. A multi-gene sequentially edited Ganoderma lucidum strain, Ganoderma lucidum H1-Δ4785Δ10331, based on a marker recovery system, was deposited at the China Center for Type Culture Collection (CCTCC) on November 27, 2025, with accession number CCTCC M 20252712.

2. A method for preparing the multi-gene sequentially edited Ganoderma lucidum strain as described in claim 1, characterized in that... Includes the following steps: Step (1): Design and preparation of sgRNA Using the CDS regions of the target genes 0Z_04785 and 0Z_10331, with sequences as described in SEQ ID NO.1 and SEQ ID NO.8 respectively, sgRNAs were designed, and suitable sgRNAs were screened using the online website http: / / crispor.tefor.net / . The target sequence is defined as the 20 bp starting 161 bp downstream of the start codon of the 0Z_04785 gene, the target sequence is defined as the 20 bp starting 419 bp downstream of the start codon of the 0Z_04785 gene, the target sequence is defined as the 20 bp starting 174 bp downstream of the start codon of the 0Z_10331 gene, and the target sequence is defined as the 20 bp starting 162 bp downstream of the start codon of the 0Z_10331 gene. The target site is located between the third and fourth bases of the target sequence; sgRNA containing the T7 promoter is amplified using primers sgRNA-F: TTCTAATACGACTCACTATA and sgRNA-R: AAAAGCACCGACTCGGT, where the sgRNA sequences of target site 1 (0Z_04785), target site 2 (0Z_04785), target site 1 (0Z_10331), and target site 2 (0Z_10331) are as described in SEQ ID NO.24-27, respectively; Step (2): Preparation of RNP The RNP complexes were assembled separately to form RNP complexes, which were named RNP-4785-1, RNP-4785-2, RNP-10331-1, and RNP-10331-2, respectively. Step (3): Preparation of donor DNA The donor DNA sequence for target 1 of gene 0Z_04785 is shown in SEQ ID NO.16, the donor DNA sequence for target 2 of gene 0Z_04785 is shown in SEQ ID NO.17, the donor DNA sequence for target 1 of gene 0Z_10331 is shown in SEQ ID NO.18, and the donor DNA sequence for target 2 of gene 0Z_10331 is shown in SEQ ID NO.

19. The donor DNA consists of four parts: the homologous upper arm, the marker gene ura3 expression cassette, the loop arm, and the homologous lower arm; the homologous upper arm is the 500 bp sequence upstream of the target site, the homologous lower arm is the 500 bp sequence downstream of the target site, and the loop arm is the 500 bp sequence upstream of the homologous upper arm. The homologous upper arm and ura3 were ligated together by overlap PCR and named as follows: 4785-1-UU / 4785-2-UU / 10331-1-UU / 10331-2-UU, which is long fragment 1; the loop arm and homologous lower arm were ligated together and named as follows: 4785-1-DD / 4785-2-DD / 10331-1-DD / 10331-2-DD, which is long fragment 2; Step (4): PEG-mediated transformation of Ganoderma lucidum protoplasts Protoplasts of strain L1-△ura3 were prepared by activation and liquid culture of PDAU and YMGU media, respectively; the protoplasts were then resuspended in STC and diluted to 10⁻⁶. 7 100μL -1 PEG-mediated transformation was performed to obtain single colonies; Step (5): Screening and verification of the 0Z_04785 edited strain Several single colonies were obtained from screening plates of strain L1-Δura3 for different target sites, and 24 colonies were randomly selected for identification. The fragments amplified in the genome of wild-type strain L1 using primers 4785-1-F / R and 4785-2-F / R were 1957bp and 1961bp in length, respectively, and the fragments amplified in the genome of the transformant were 4857bp and 4858bp in length, respectively. According to agarose gel electrophoresis, all three transformants were found to have completely recovered the ura3 selection marker, including one Δ4785-1 strain with target 1 edited, and two Δ4785-2 and Δ4785-3 strains with target 2 edited. Step (6): Recycling of filter tags Protoplasts were prepared from the positive transformants Δ4785-1, Δ4785-2, and Δ4785-3 with complete ura3 gene expression cassette filling. An appropriate amount of protoplast dilution was evenly spread on YMGAU medium containing 400 mg / L 5-FOA and cultured. Several single colonies were obtained from each. Ten colonies from each were randomly selected and transferred to PDAU medium containing 400 mg / L 5-FOA for secondary screening. Edge hyphae were picked and inoculated onto PDA medium for propagation. Genomic DNA of the transformants was extracted. Using primers 4785-1-F / R and 4785-2-F / R, fragments of 1957 bp and 1961 bp were amplified in the genome of the wild-type strain L1, respectively, and fragments of 1337 bp and 1338 bp were amplified in the genome of the transformant, respectively. Based on the agarose gel electrophoresis results, six edited strains were obtained. Four strains were obtained using Δ4785-1 as the test strain: Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, and Δ4785Δura3-4; one strain was obtained using Δ4785-2 as the test strain: Δ4785Δura3-5; and one strain was obtained using Δ4785-3 as the test strain: Δ4785Δura3-6. Step (7): Editing of the 0Z_10331 gene in the 0Z_04785 gene-edited strain The strains Δ4785Δura3-1, Δ4785Δura3-2, Δ4785Δura3-3, Δ4785Δura3-4, Δ4785Δura3-5 and Δ4785Δura3-6 were activated and cultured in liquid medium using PDAU and YMGU, respectively, to prepare protoplasts for PEG-mediated transformation. Using primers 10331-1 / 2-F / R, fragments of 2221 bp were amplified in the genome of the wild-type strain L1, and fragments of 5118 bp were amplified in the genome of the transformants. Agarose gel electrophoresis results showed that the PCR amplification product band size of one of the transformants was consistent with the expectation. Sequencing analysis showed that the transformant completely restored the ura3 selection marker, and it was named H1-Δ4785Δ10331. H1-Δ4785Δ10331 is the desired Ganoderma lucidum strain with continuous multi-gene editing. The strain was deposited at the China Center for Type Culture Collection (CCTCC) on November 27, 2025, with accession number CCTCM20252712.

3. The application of the multi-gene sequentially edited Ganoderma lucidum strain based on the marker recovery system as described in claim 1 in breeding.

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