Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing
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 synthesize...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-10-01
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| Series: | Cell & Bioscience |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13578-019-0350-7 |
| _version_ | 1818974191427256320 |
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| author | Zheng Hu Li Wang Zhaoying Shi Jing Jiang Xiangning Li Yonglong Chen Kai Li Dixian Luo |
| author_facet | Zheng Hu Li Wang Zhaoying Shi Jing Jiang Xiangning Li Yonglong Chen Kai Li Dixian Luo |
| author_sort | Zheng Hu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T15:36:08Z |
| format | Article |
| id | doaj.art-28a79de7740d499f9f6ced4a15a690ce |
| institution | Directory Open Access Journal |
| issn | 2045-3701 |
| language | English |
| last_indexed | 2024-12-20T15:36:08Z |
| publishDate | 2019-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell & Bioscience |
| spelling | doaj.art-28a79de7740d499f9f6ced4a15a690ce2022-12-21T19:35:25ZengBMCCell & Bioscience2045-37012019-10-01911710.1186/s13578-019-0350-7Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editingZheng Hu0Li Wang1Zhaoying Shi2Jing Jiang3Xiangning Li4Yonglong Chen5Kai Li6Dixian Luo7The First People’s Hospital of Chenzhou, Affiliated to University of Southern Medical UniversityThe First People’s Hospital of Chenzhou, Affiliated to University of Southern Medical UniversityDepartment of Biology, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and TechnologyNational & Local Joint Engineering Laboratory for High-throughput Molecular Diagnosis Technology, Affiliated to The First People’s Hospital of Chenzhou, University of South ChinaNational & Local Joint Engineering Laboratory for High-throughput Molecular Diagnosis Technology, Affiliated to The First People’s Hospital of Chenzhou, University of South ChinaDepartment of Biology, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and TechnologyNational & Local Joint Engineering Laboratory for High-throughput Molecular Diagnosis Technology, Affiliated to The First People’s Hospital of Chenzhou, University of South ChinaThe First People’s Hospital of Chenzhou, Affiliated to University of Southern Medical UniversityAbstract 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.http://link.springer.com/article/10.1186/s13578-019-0350-7Overlap extension PCRMultiple overlapping primerssgRNATranscription templateCas9 nuclease |
| spellingShingle | 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 Cell & Bioscience Overlap extension PCR Multiple overlapping primers sgRNA Transcription template Cas9 nuclease |
| title | Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing |
| title_full | Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing |
| title_fullStr | Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing |
| title_full_unstemmed | Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing |
| title_short | Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing |
| title_sort | customized one step preparation of sgrna transcription templates via overlapping pcr using short primers and its application in vitro and in vivo gene editing |
| topic | Overlap extension PCR Multiple overlapping primers sgRNA Transcription template Cas9 nuclease |
| url | http://link.springer.com/article/10.1186/s13578-019-0350-7 |
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