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Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

Nature Communications. 2017; 
Serif Senturk, Nitin H. Shirole, Dawid G. Nowak, Vincenzo Corbo, Debjani Pal, Alexander Vaughan, David A. Tuveson, Lloyd C. Trotman, Justin B. Kinney & Raffaella Sordella
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Codon Optimization  Here, Cre-ERT2 protein codon was optimized for human expression with three naturally existing BsmBI restriction enzyme sites removed by silent mutations was synthesized, cloned into pUC57 cloning vector and sequence verified by Genscript. Get A Quote

摘要

The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with ... More

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