图2. 靶向随机突变的实验流程及sgRNA靶点库设计示例
(A) 靶向随机突变水稻基因的实验流程; (B) sgRNA靶点库设计示例。确定目标基因靶向随机突变区域为第3、第4外显子E3和E4 (270和330 bp), 设计60个sgRNA靶点并分为4组。其中组1、组2靶点PAM序列分别为NGD和HCN, 可由引物E3-F/E3-R扩增获得长度约700 bp的片段, 以便后续使用T7E1酶切法检测突变体植株; 组3、组4靶点PAM序列分别为NGD和HCN, 可由引物E4-F/E4-R扩增获得长度约700 bp的片段, 以便后续使用T7E1酶切法检测突变体植株。

Figure 2. Schematic of the procedure for targeted random mutagenesis and illustration of design for sgRNA pool
(A) Schematic of the procedure for targeted random mutagenesis; (B) Illustration of design for sgRNA pool. Chose the target region of 3^rd and 4^th exons (E3: 270 bp and E4: 330 bp), then design 60 sgRNAs divided into 4 groups. sgRNAs in group 1 and 2 target NGD and HCN PAM sequences and the target region (700 bp) can be amplified using primers E3-F/E3-R. sgRNAs in group 3 and 4 target NGD and HCN PAM sequences and the target region (700 bp) can be amplified using primers E4-F/E4-R. Amplicons are about 700 bp and suitable for T7E1 assay.