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

10.1186/s13578-019-0350-7

Cell and Bioscience   影响因子和分区

2045-3701

2045-3701

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.

Overlap extension PCR Multiple overlapping primers sgRNA Transcription template Cas9 nuclease

SCI

英语

其他

Key Project of the First People's Hospital of Chenzhou[N2019-001]

Biochemistry & Molecular Biology

Biochemistry & Molecular Biology

WOS:000492859400001

BMC

Web of Science

1.Affiliated Univ Southern Med Univ, Peoples Hosp Chenzhou 1, Chenzhou 423000, Peoples R China
2.Univ South China, Affiliated Peoples Hosp Chenzhou 1, Natl & Local Joint Engn Lab High Throughput Mol D, Chenzhou 423000, Peoples R China
3.Univ South China, Translat Med Inst, Chenzhou 423000, Peoples R China
4.Southern Univ Sci & Technol, Guangdong Prov Key Lab Cell Microenvironm & Dis R, Shenzhen Key Lab Cell Microenvironm, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China
5.Genetalks Biotech Changsha Ltd Liabil Co, Changsha 410013, Hunan, Peoples R 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[J]. Cell and Bioscience,2019,9(1).

Hu, Zheng.,Wang, Li.,Shi, Zhaoying.,Jiang, Jing.,Li, Xiangning.,...&Luo, Dixian.(2019).Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing.Cell and Bioscience,9(1).

Hu, Zheng,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".Cell and Bioscience 9.1(2019).