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A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo

Serif Senturk, Nitin H. Shirole, Dawid D. Nowak, Vincenzo Corbo, Alexander Vaughan, David A. Tuveson, Lloyd C. Trotman, Adam Kepecs, Frank Stegmeier, Raffaella Sordella
doi: https://doi.org/10.1101/023366
Serif Senturk
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Nitin H. Shirole
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Dawid D. Nowak
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Vincenzo Corbo
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Alexander Vaughan
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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David A. Tuveson
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Lloyd C. Trotman
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Adam Kepecs
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Frank Stegmeier
2Novartis Institute for BioMedical Research 250 Massachusetts Avenue, 3C-302 Cambridge, MA 02139
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Raffaella Sordella
1Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
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Abstract

The Cas9/CRISPR system is a powerful tool for studying gene function. Here we describe a method that allows temporal control of Cas9/CRISPER activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-CAS9) enables conditional Cas9 expression in vitro in the presence of an FKBP12 synthetic ligand and temporal control of gene-editing. Further, we show that this strategy can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest, without the latter being co-modulated. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic identification of essential genes and the interrogation of genes functional interactions.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 28, 2015.
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A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo
Serif Senturk, Nitin H. Shirole, Dawid D. Nowak, Vincenzo Corbo, Alexander Vaughan, David A. Tuveson, Lloyd C. Trotman, Adam Kepecs, Frank Stegmeier, Raffaella Sordella
bioRxiv 023366; doi: https://doi.org/10.1101/023366
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A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo
Serif Senturk, Nitin H. Shirole, Dawid D. Nowak, Vincenzo Corbo, Alexander Vaughan, David A. Tuveson, Lloyd C. Trotman, Adam Kepecs, Frank Stegmeier, Raffaella Sordella
bioRxiv 023366; doi: https://doi.org/10.1101/023366

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