基于优化sgRNA系统提高海岛棉CRISPR/Cas9基因组编辑功效的研究

李继洋,胡燕,姚瑞,代培红,刘晓东

Enhancing CRISPR/Cas9 genomic editing efficiency based on optimization of sgRNA of Gossypium barbadense L.

Ji-Yang LI,Yan HU,Rui YAO,Pei-Hong DAI,Xiao-Dong LIU

图5 海岛棉转化不同靶序列编辑效应检测结果 A: 1为转化GbU6-5P-GGB-sgRNA1-Cas9I载体酶切前直接PCR扩增结果, 2为转化GbU6-5P-GGB-sgRNA1-Cas9I载体酶切后PCR扩增结果, 3为转化GbU6-5P-sgRNA1-Cas9I载体酶切前直接PCR扩增结果, 4为转化GbU6-5P-sgRNA1-Cas9I载体酶切后PCR扩增结果。B: 1为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I直接PCR扩增结果, 2为转化GbU6-5P-sgRNA1-GbU6-4P-sgRNA2- Cas9I直接PCR扩增结果。C: G1、E1为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I酶切前直接PCR扩增结果, G2、E2为转化GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I酶切后PCR扩增结果, G3、E3为转化GbU6-5P-sgRNA1-GbU6-4P- sgRNA2-Cas9I酶切前直接PCR扩增结果, G4、E4为转化GbU6-5P-sgRNA1-GbU6-4P-sgRNA2-Cas9I酶切后PCR扩增结果。M1为1 kb plus marker, M2为2 kb plus II marker。

Fig. 5 Transformation of different target sequence editing effect test results of Gossypium barbadense L. A: 1 is the result of direct PCR before transformation of GbU6-5P-GGB-sgRNA1-Cas9I vector, 2 is PCR amplification after transformation of GbU6-5P-GGB-sgRNA1-Cas9I vector As a result, 3 was obtained by direct PCR amplification before transformation of GbU6-5P-sgRNA1- Cas9I vector and 4 was PCR amplification after transformation with GbU6-5P-sgRNA1-Cas9I vector. B: 1 was transformed with GbU6- 5P::GGB-sgRNA1-GbU6-4P::GGB-sgRNA2-Cas9I direct PCR amplification results, 2 for the transformation of GbU6-5P-sgRNA1-GbU6- 4P-sgRNA2-Cas9I direct PCR amplification results. C: G1 and E1 are the results of direct PCR before transformation of GbU6-5P:: GGB-sgRNA1-GbU6-4P::ERA1-sgRNA2-Cas9I, G2 and E2 are the results of transformation of GbU6-5P::GGB-sgRNA1-GbU6-4P::ERA1- sgRNA2-Cas9 digestion after PCR amplification results, G3 and E3 to transform GbU6-5P-sgRNA1-GbU6-4P-sgRNA2-Cas9I digestion before direct PCR amplification results, G4 and E4 to transform GbU6-5P-sgRNA1-PCR amplification results after GbU6-4P-sgRNA2-Cas9 digestion. M1 is 1 kb plus marker, M2 is 2 kb plus II marker.