棉花CRISPR/Cas9基因编辑有效sgRNA高效筛选体系的研究

周冠彤, 雷建峰, 代培红, 刘超, 李月, 刘晓东

Efficient screening system of effective sgRNA for cotton CRISPR/Cas9 gene editing

ZHOU Guan-Tong, LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong

图2 GhU6-5P::MAPKKK2-sgRNA靶向突变的检测 A: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-Cas9单株样品基因组PCR扩增产物酶切后结果。B: M: 2K plus II DNA marker; 1、2分别为对照载体基因组PCR扩增产物酶切前后结果; 3~6为转化GhU6-5P::MAPKKK2-sgRNA-1300单株样品基因组PCR扩增产物酶切后结果。C: GhMAPKKK2-sgRNA靶序列及突变PCR产物的测序。M1发生在D亚组的GhMAPKKK2, M2、M3发生在A亚组GhMAPKKK2。D: 突变PCR产物克隆的测序峰图, 红色方框指示碱基相比对照发生突变区域。PCR/RE表示PCR扩增产物酶切分析。

Fig. 2 GhU6-5P::MAPKKK2-sgRNA targeted mutations A: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and after the enzyme digestion; 3-6: PCR/RE assay of GhU6-5P::MAPKKK2-sgRNA-Cas9 genome of single plant sample after enzyme digestion. B: M: 2K plus II DNA marker; 1, 2: PCR/RE assay of negative control genome before and of single plant sample after the enzyme digestion; 3-6: PCR/RE assay of GhU6-5P::MAPKKK2-sgRNA-1300 genome after enzyme digestion. C: sequencing of GhMAPKKK2-sgRNA target sequence and mutant PCR products. M1 occurred at GhMAPKKK2 from D subgenome, M2, M3 occurred at GhMAPKKK2 from A subgenome. D: sequencing peak diagram of the mutant PCR product clone, the red box indicates that the base has a mutation region compared to the control. PCR/RE indicates PCR/restriction enzyme assays.