图1 基因编辑元件结构及编辑过程示意图A：左侧从上至下分别表示cas9表达盒、不同启动子的sgRNA表达盒及donor DNA的组成。Cas9表达盒由Pterf启动子、加有核定位信号肽NLS的编码序列及Tterf终止子组成；sgRNA的启动子分别使用T7启动子、5SrRNA启动子（包括134 bp和395 bp两种长度）、tRNAGly15启动子、tRNAGly17启动子；右侧上方蓝色虚线框中为正常sgRNA的tracrRNA骨架，下方虚线框内的sgRNA tracrRNA骨架携带有一个发夹结构以锁型图表示；Donor DNA由上下游各500 bp的同源臂和中间的潮霉素抗性基因组成。B：CRISPR/Cas9系统工作的简略示意图，图中展示了表达盒法和RNP法所需的编辑原件。以表达盒法进行转化时，需将Cas9表达盒和sgRNA 表达盒连同Donor DNA一起加入制备好的原生质体中；RNP法进行转化需将Cas9蛋白与sgRNA体外孵育后以RNP复合体的形式与Donor DNA一起加入制备好的原生质体中进行转化。目标基因fwnA被剪切，剪切位点两侧基因组序列与Donor DNA的同源臂发生同源重组，使潮霉素基因插入fwnA基因内部，使基因失活，导致孢子颜色由黑变白，基因检测引物标注在Donor DNA的示意图上

Fig. 1 Schematic diagram of gene editing element structure and editing processA: The left side from top to bottom indicates the composition of cas9 expression cassette, sgRNA expression cassette of different promoters and donor DNA, respectively. The Cas9 expression cassette is composed of Pterf promoter, coding sequence, nuclear localization signal peptide NLS and Tterf terminator. The promoters of sgRNA are T7 promoter, 5SrRNA promoter (including 134 bp and 395 bp), tRNAGly15 promoter and tRNAGly17 promoter, respectively. The tracrRNA skeleton of normal sgRNA is in the blue dotted box above the right side, and the sgRNA tracrRNA skeleton in the dotted box below carries a hairpin structure represented by a “lock” graph. Donor DNA consists of 500 bp upstream and downstream homologous arms and intermediate hygromycin resistance genes. B: A brief schematic diagram of the CRISPR/Cas9 system, which shows the editing originals required for the expression cassette method and the RNP method. When the expression cassette method is used for transformation, the Cas9 expression cassette and sgRNA expression cassette need to be added to the prepared protoplasts together with Donor DNA. When the RNP method was used for transformation, Cas9 protein and sgRNA were incubated in vitro, and the two were added to the prepared protoplasts together with Donor DNA in the form of RNP complex for transformation. The target gene fwnA was cut, and the genome sequences on both sides of the cutting site were homologously recombined with the homologous arm of Donor DNA, thus the hygromycin gene was inserted into the fwnA gene, and the gene was inactivated, resulting in the spore color from black to white, and the gene detection primers were labeled on the schematic diagram of Donor DNA

图2 Cas9、sgRNA表达盒及Donor DNA的扩增1：以134 bp 5S rRNA 为启动子的sgRNA；2：以395 bp 5S rRNA为启动子的sgRNA；3：以tRNAGly17为启动子的sgRNA；4：以tRNAGly15为启动子的sgRNA；5：tRNAGly15为启动子且融合了“lock”结构的sgRNA；6：供体DNA；7：Cas9表达盒

Fig. 2 Amplification of Cas9 expression cassette, sgRNA expression cassette and Donor DNA1: sgRNA with 134 bp 5S rRNA as promoter; 2: sgRNA with 395 bp 5S rRNA as promoter; 3: a sgRNA with tRNAGly17 as the promoter; 4: a sgRNA with tRNAGly15 as the promoter; 5: sgRNA with tRNAGly15 as promoter and fused with “lock” structure; 6: Donor DNA; 7:Cas9 expression cassette

图3 表达盒法中转化子生长情况及不同sgRNA启动子的基因编辑效率A：分别为5S rRNA 134 bp和tRNAGly15两个实验组转化子的生长情况（黑孢、白孢、杂孢颜色如图所示）； B：不同长度的5S启动子、tRNAGly15启动子、tRNAGly17启动子的sgRNA的基因编辑效率

Fig. 3 Growth of transformants and the gene editing efficiency of different sgRNA promoters during transformation by expression cassette methodA: The growths of transformants in the two experimental groups of 5S rRNA 134 bp and tRNAGly15 (the colors of black spores, white spores, and miscellaneous spores are shown in the figure). B: The gene editing efficiency of sgRNA with different lengths of 5S promoter, tRNAGly15 promoter and tRNAGly17 promoter

图4 RNP法中“lock”结构的sgRNA的基因编辑效率图中为使用RNP法时正常sgRNA结构与融合了“lock”结构的sgRNA的基因编辑效率比较。每个实验组进行3次生物学重复，数据用spss软件进行误差分析。误差线代表标准误的大小；不同字母之间的显著性水平为0.05（下同）

Fig. 4 Gene editing efficiency of sgRNA with “lock” structure in RNP methodThe histogram in Fig.4 shows the gene editing efficiency of sgRNA containing ordinary sgRNA structure and sgRNA fused with “lock” structure.Three biological replicates were performed in each experimental group, and the obtained data were analyzed by spss software. The error line indicates the size of the standard error.The significance level between different letters is 0.05 ( the same below )

图 5 表达盒法中 tRNAGly15启动子叠加“lock”结构的基因编辑效率以tRNAGly15为启动子的sgRNA与以tRNAGly15为启动子并融合“lock”结构的sgRNA的基因编辑效率

Fig. 5 Gene editing efficiency of tRNAGly15 promoter superimposed with “lock” structure in expression cassette methodThe gene editing efficiency of sgRNA with tRNAGly15 as the promoter and sgRNA with tRNAGly15 as the promoter and fused with “lock” structure