Abstract 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.

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overlap extension polymerase chain reaction pcr dna assembly sgrna

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

Zheng Hu,Li Wang,Zhaoying Shi,Jing Jiang,Xiangning Li,Yonglong Chen,Kai Li,Dixian Luo,"Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing" 9

Zheng Hu,Li Wang,Zhaoying Shi,Jing Jiang,Xiangning Li,Yonglong Chen,Kai Li,Dixian Luo,"Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing"