Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing

Hu，Zheng1,2,3; Wang，Li1,2,3; Shi，Zhaoying4; Jiang，Jing2,3; Li，Xiangning2,3; Chen，Yonglong4; Li，Kai2,3,5; Luo，Dixian1,2,3

2019-10-24

Cas9 nuclease Multiple overlapping primers Overlap extension PCR sgRNA Transcription template

英语

Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

10.1186/s13578-019-0350-7

Cell and Bioscience

9

1

SCI

其他

2-s2.0-85074460542

Scopus

WOS:000492859400001

2045-3701

1.First People's Hospital of Chenzhou,Affiliated to University of Southern Medical University,Chenzhou,423000,China
2.National and Local Joint Engineering Laboratory for High-throughput Molecular Diagnosis Technology,Affiliated to the First People's Hospital of Chenzhou,University of South China,Chenzhou,423000,China
3.Translational Medicine Institute,University of South China,Chenzhou,423000,China
4.Department of Biology,Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research,Shenzhen Key Laboratory of Cell Microenvironment,Southern University of Science and Technology,Shenzhen, Guangdong,518055,China
5.Genetalks Bio-tech (Changsha) Limited Liability Company,Changsha, Hunan,410013,China

Hu，Zheng,Wang，Li,Shi，Zhaoying,et al. Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing. 2019-10-24.