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

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成果类型：

期刊论文

作者：

Hu, Zheng*;Wang, Li;Shi, Zhaoying;Jiang, Jing;Li, Xiangning;...

通讯作者：

Hu, Zheng;Luo, Dixian;Li, Kai

作者机构：

[Hu, Z; Luo, DX; Wang, Li; Hu, Zheng; Luo, Dixian] Affiliated Univ Southern Med Univ, Peoples Hosp Chenzhou 1, Chenzhou 423000, Peoples R China.

[Jiang, Jing; Li, Kai; Hu, Z; Li, K; Luo, DX; Wang, Li; Hu, Zheng; Luo, Dixian; Li, Xiangning] Univ South China, Affiliated Peoples Hosp Chenzhou 1, Natl & Local Joint Engn Lab High Throughput Mol D, Chenzhou 423000, Peoples R China.

[Jiang, Jing; Li, Kai; Hu, Z; Li, K; Luo, DX; Wang, Li; Hu, Zheng; Luo, Dixian; Li, Xiangning] Univ South China, Translat Med Inst, Chenzhou 423000, Peoples R China.

[Shi, Zhaoying; Chen, Yonglong] 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.

[Li, Kai] Genetalks Biotech Changsha Ltd Liabil Co, Changsha 410013, Hunan, Peoples R China.

通讯机构：

[Hu, Z; Luo, DX] A

[Hu, Z; Li, K; Luo, DX] U

[Li, Kai] G

Affiliated Univ Southern Med Univ, Peoples Hosp Chenzhou 1, Chenzhou 423000, Peoples R China.

Univ South China, Affiliated Peoples Hosp Chenzhou 1, Natl & Local Joint Engn Lab High Throughput Mol D, Chenzhou 423000, Peoples R China.

语种：

英文

关键词：

Overlap extension PCR;Multiple overlapping primers;sgRNA;Transcription template;Cas9 nuclease

期刊：

Cell and Bioscience

ISSN：

2045-3701

年：

2019

卷：

期：

页码：

1-7

DOI：

10.1186/s13578-019-0350-7

基金类别：

This work was partly supported by the Hunan Province Science Fund for Distinguished Young Scholars (2018JJ1021), the Key R&D Program of Hunan Province (2017SK2172), the Natural Science Foundation of Hunan Province (2017JJ2004), the Health Department Project of Hunan Province (B20180378, C20180166,C2019003), the Science and Technique Foundation of Chenzhou (jsyf2017021, zdyf201838), the Key Project of the First People’s Hospital of Chenzhou (N2019-001) and the 2018’s Changsha Municipal Project for Overseas Experts (2018).

机构署名：

本校为通讯机构

院系归属：

医学院

摘要：

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 s...

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

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