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bioRxiv · 10.1101/027664

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER), a novel method for selective transformation

Lynn J. Rothschild·Daniel T. Greenberg·Jack R. Takahashi·Kirsten A. Thompson·Akshay J. Maheshwari·Ryan E. Kent·Griffin McCutcheon·Joseph D. Shih·Charles L. Calvet·Tyler D. Devlin·Tina Ju·Daniel P. Kunin·Erica K. Lieberman·Thai Nguyen·Forrest G. Tran·Daniel Z. Xiang·Kosuke Fujishima
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Abstract

The CRISPR/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be applied to facilitate efficient plasmid selection for transformation as well as selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts after restriction enzyme digestion and ligation. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes unwanted ligation byproducts and increases transformation efficiency of desired inserts from 20% up to 97% {+/-} 3%. This CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient method for obtaining specific cloning products.

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Lynn J. Rothschild, Daniel T. Greenberg, Jack R. Takahashi, Kirsten A. Thompson, Akshay J. Maheshwari, Ryan E. Kent, Griffin McCutcheon, Joseph D. Shih, Charles L. Calvet, Tyler D. Devlin, Tina Ju, Daniel P. Kunin, Erica K. Lieberman, Thai Nguyen, Forrest G. Tran, Daniel Z. Xiang, Kosuke Fujishima. 2015-09-27. CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER), a novel method for selective transformation. https://doi.org/10.1101/027664

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