Method for improving protoplast conversion efficiency or gene editing efficiency of fungi and kit
A protoplast transformation and gene editing technology, applied in the biological field, can solve the problems of PAM dependence, off-target, low genome editing efficiency, etc.
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Embodiment 1
[0068] Example 1, sgRNA in vitro transcription and purification
[0069] For the target gene, design the sgRNA recognition site, and synthesize the following primers for forming sgRNA: sgRNA-F: TAATACGACTCACTATA-sgRNA site-GTTTTAGGCTAGAAAT AGCAA.
[0070] The sgRNA-F sequence designed for Trichoderma reesei RutC30 orotate phosphoribosyltransferase ura5 (SEQ ID NO: 1) is:
[0071] sgRNA-Trura5-F: TAATACGACTCACTATA GGCGAGGGCGGCAACATCGT GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO:5).
[0072] The sgRNA-F sequence designed for ura5 (SEQ ID NO:2) of Cordyceps militaris Cm01 is:
[0073] sgRNA-Cmura5-F: TAATACGACTCACTATA GGACCGGATGGAGAAGCTTC GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 6).
[0074] The sgRNA-F sequence designed for the polyketide synthase wA of Aspergillus oryzae NSAR1 is:
[0075] sgRNA-wA-F: TAATACGACTCACTATA GGGAAGATCTTCCGTTCAGA GTTTTAGAGCTAGAAATAGCAA (SEQ ID NO: 7).
[0076] These sgRNA-F primers were PCR amplified with sgRNA-R primers respectively, and the sgRNA template...
Embodiment 2
[0080] Example 2, sgRNA-Cas9 complex assembled in vitro
[0081] The purified sgRNA and commercialized Cas9 (novoprotein E365-01A) were added to the reaction buffer at a ratio of 70:1 (mole), and the reaction buffer was 2× protoplast solution 2 (2M sorbitol, 100mM CaCl 2 , 2MTris-HCl, pH 7.5) and 10×active buffer (200mM HEPES free acid, 1.5M KCl, 5mM DTT, 1mM EDTA, 100mM MgCl 2 ·6H 2 O, pH 7.5), mix well and incubate at 37°C for 15min.
[0082] sgRNA-Cas9 assembly method of Trichoderma reesei ura5 gene: Add the purified sgRNA-Trura5 and Cas9 into the reaction system at a ratio of 70:1, and incubate at 37°C for 15 minutes. And by controlling the addition of sgRNA and Cas9, 20nM, 60nM, 100nM, 180nM sgRNA-Cas9 complexes were prepared respectively.
[0083] The sgRNA-Cas9 assembly method of the Cordyceps militaris ura5 gene: add the purified sgRNA-Cmura5 and Cas9 at a ratio of 70:1 (6ug for Cas9) into the reaction system, and incubate at 37°C for 15min.
[0084] The sgRNA-Cas9...
Embodiment 3
[0085] Example 3, adding TritonX-100 to improve the efficiency of transformation and gene editing
[0086] Using Trichoderma reesei Rut C30 as the chassis cell, the orotate phosphoribosyltransferase ura5 gene (SEQ ID NO: 1; the 355th to 374th position is the sgRNA target sequence) was mutated by double in vitro CRISPR / Cas9 technology. The sgRNA-Cas9 complex (the sgRNA template is formed by PCR amplification of sgRNA-Trura5-F and sgRNA-R) was transformed, and the method of increasing the number of transformants obtained by gene transformation and improving the efficiency of gene editing was studied.
[0087] The preparation of protoplasts was modified according to the literature (Rantasalo, A. et al. A universal gene expression system for fungi. Nucleic Acids Res, 2018 46, e111), and the hyphae of protoplasts were prepared by culturing in cellophane-lined PDA at 28°C for 15 hours To obtain, the concentration of lyase (sigma) was 5 mg / ml.
[0088] Transformation system: collect...
PUM
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