甘蓝型油菜和甘蓝CRISPR/Cas9编辑效果的快速检测

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

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的成功率,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Bna C. WRKY11. a基因设计了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Bol PDS3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,可用于CRISPR/Cas9的编辑效果快速检测。

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/sg RNA 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,which 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.