A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

被引:11
|
作者
Liu, Xiaoli [1 ]
Zhou, Xiujuan [1 ]
Li, Kang [2 ,3 ]
Wang, Dehong [2 ,3 ]
Ding, Yuanhao [1 ]
Liu, Xianqing [1 ]
Luo, Jie [1 ,2 ,3 ]
Fang, Chuanying [1 ]
机构
[1] Hainan Univ, Coll Trop Crops, Haikou, Hainan, Peoples R China
[2] Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan, Hubei, Peoples R China
[3] Huazhong Agr Univ, Natl Ctr Plant Gene Res, Wuhan, Hubei, Peoples R China
来源
PEERJ | 2020年 / 8卷
基金
中国国家自然科学基金;
关键词
Cloning system; Multiplex PCR; CRISPR/Cas9; Genome editing; Rice; FUNCTIONAL GENOMICS; MUTAGENESIS; OUTGROWTH; TOOL;
D O I
10.7717/peerj.8491
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Rapidly growing genetics and bioinformatics studies provide us with an opportunity to obtain a global view of the genetic basis of traits, but also give a challenge to the function validation of candidate genes. CRISPR/Cas9 is an emerging and efficient tool for genome editing. To construct expression clones for the CRISPR/Cas9, most current methods depend on traditional cloning using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on multiple steps of PCR. We developed a system for introducing sgRNA expression cassette(s) directly into plant binary vectors in one step. In this system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR took place. Whilst, two sgRNA expression cassettes were amplified in a single round of PCR. Subsequently, an LR or Golden gate reaction was set up with unpurified PCR product and befitting destination vector. We are able to construct expression clones within 36 h, which greatly improves efficiency and saves cost. Furthermore, the efficiency of this system was verified by an agrobacterium-mediated genetic transformation in rice. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing.
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页数:16
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