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タイトルCRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation
本文(外部サイト)http://hdl.handle.net/2060/20160004668
著者(英)Shih, Joseph D.; Thompson, Kirsten A.; Greenberg, Daniel T.; Rothschild, Lynn J.; Takahashi, Jack R.; Lieberman, Erica; Calvet, Charles; Kent, Ryan E.; McCutcheon, Griffin; Nguyen, Thai; Fujishima, Kosuke; Ju, Tina; Kunin, Daniel; Maheshwari, Akshay J.; Xiang, Daniel; Tran, Forrest; Devlin, Tyler D.
著者所属(英)NASA Ames Research Center
発行日2015-09-27
言語eng
内容記述The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation.
NASA分類Life Sciences (General)
レポートNOARC-E-DAA-TN29411
権利Copyright, Distribution as joint owner in the copyright


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