Optimized Plasmid Construction Strategy for Cas9
Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and att...
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Cell Physiol Biochem Press GmbH & Co KG
2018-07-01
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doaj-ebe543bf676a425c8db449a1b37d0f012020-11-25T01:57:03ZengCell Physiol Biochem Press GmbH & Co KGCellular Physiology and Biochemistry1015-89871421-97782018-07-0148113113710.1159/000491669491669Optimized Plasmid Construction Strategy for Cas9Jianyong XuWenlei LiMD. Munnaf HossenYuning JiaLingyun LiZhong HuangBackground/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.https://www.karger.com/Article/FullText/491669RNA guided endonucleaseGenome editingCas9Guide RNAgRNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jianyong Xu Wenlei Li MD. Munnaf Hossen Yuning Jia Lingyun Li Zhong Huang |
spellingShingle |
Jianyong Xu Wenlei Li MD. Munnaf Hossen Yuning Jia Lingyun Li Zhong Huang Optimized Plasmid Construction Strategy for Cas9 Cellular Physiology and Biochemistry RNA guided endonuclease Genome editing Cas9 Guide RNA gRNA |
author_facet |
Jianyong Xu Wenlei Li MD. Munnaf Hossen Yuning Jia Lingyun Li Zhong Huang |
author_sort |
Jianyong Xu |
title |
Optimized Plasmid Construction Strategy for Cas9 |
title_short |
Optimized Plasmid Construction Strategy for Cas9 |
title_full |
Optimized Plasmid Construction Strategy for Cas9 |
title_fullStr |
Optimized Plasmid Construction Strategy for Cas9 |
title_full_unstemmed |
Optimized Plasmid Construction Strategy for Cas9 |
title_sort |
optimized plasmid construction strategy for cas9 |
publisher |
Cell Physiol Biochem Press GmbH & Co KG |
series |
Cellular Physiology and Biochemistry |
issn |
1015-8987 1421-9778 |
publishDate |
2018-07-01 |
description |
Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA. |
topic |
RNA guided endonuclease Genome editing Cas9 Guide RNA gRNA |
url |
https://www.karger.com/Article/FullText/491669 |
work_keys_str_mv |
AT jianyongxu optimizedplasmidconstructionstrategyforcas9 AT wenleili optimizedplasmidconstructionstrategyforcas9 AT mdmunnafhossen optimizedplasmidconstructionstrategyforcas9 AT yuningjia optimizedplasmidconstructionstrategyforcas9 AT lingyunli optimizedplasmidconstructionstrategyforcas9 AT zhonghuang optimizedplasmidconstructionstrategyforcas9 |
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1724976679154089984 |