甘蓝型油菜和甘蓝CRISPR/Cas9编辑效果的快速检测

CRISPR/Cas9系统是目前最常用的基因组定点编辑工具,通过瞬时表达试验提前验证Cas9/sgRNA载体诱导的突变效率,可以提高基因组定点编辑成功的几率,且显著节省费用及时间。本研究开发了一种利用农杆菌介导的操作简单、适用性好、成本低廉的叶片瞬时表达技术,可用于快速检测甘蓝型油菜和甘蓝中CRISPR/Cas9的编辑效果。针对甘蓝型油菜BnaC.WRKY11.a设计了2个靶位点Tgt1（Target 1）和Tgt2（Target 2）,并构建了Cas9/sgRNA-Tgt1/2多重突变载体,在甘蓝型油菜叶片中瞬时表达后,2个靶位点都出现突变,突变效率达11.2%～82.2%。针对甘蓝BolPDS3基因设计了1个靶位点Tgt3（Target3）,并构建了Cas9/sgRNA-Tgt3敲除载体,在甘蓝叶片瞬时表达后,Tgt3发生了突变,且大部分为碱基缺失突变,缺失的数目为1～18bp不等,同时还存在少量碱基插入以及碱基替换等突变类型。结果表明这种甘蓝型油菜和甘蓝的叶片瞬时表达技术操作简单、适用性好、成本低廉,CRISPR/Cas9的编辑效果快速易检测。

Rapid detection of CRISPR/Cas9-mediated genome editing efficiency in Brassica napus and B. oleracea

CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated 9) system is the simplest and most commonly used targeted genome editing tool, which has shown the enormous application potential in many fields. The mutation efficiency mediated by CRISPR/Cas9 was affected by many factors, it used transient expression technique to evaluate the mutation efficiency of Cas9/sgRNA vector before generating the mutant plants, which was a favourite way to improve research efficiency. In this study, we developed an Agrobacterium-mediated transient expression technique, Wichita was easy to maneuve and cost-effective for rapid testing the efficiency of CRISPR/Cas9-mediated genome editing. Cas9/sgRNA-Tgt1/2 construction targeted BnaC.WRKY11.a was constructed to mediate multiple mutations with Tgt1 (Target1) and Tgt2 (Target2) as targets in B. napus. Multiple genome modifications were achieved in B. napus after agroinfiltration by Cas9/sgRNA-Tgt1/2 construction. The frequencies of targeted mutagenesis mediated by Tgt1 and Tgt2 were in the range of 11.2%-82.2%. Beyond that, Tgt3 (Target3) targeting to BolPDS3 was designed, Cas9/sgRNA-Tgt3 construction was generated for genome editing in B. oleracea. Transient mutation experiments showed that mutations happened in BolPDS3 at Tgt3. Most of the mutations were deletion mutations, of a range between 1-18 bp. A few of the mutations were insertion and substitution mutations. Our results showed that vacuum-assisted infiltration by Agrobacterium could be an effective transient expression method being cost-effective with easy maneuverability for B. oleracea and B. napus, it could be used for rapid detection of the mutation efficiency mediated by CRISPR/Cas9.