title: Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.
abstract: 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 single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Serif
authors.initials: S
authors.lastname: Senturk
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.|Graduate Program in Genetics, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Nitin H
authors.initials: NH
authors.lastname: Shirole
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Dawid G
authors.initials: DG
authors.lastname: Nowak
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Corbo
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.|Graduate Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Debjani
authors.initials: D
authors.lastname: Pal
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Vaughan
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Tuveson
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Lloyd C
authors.initials: LC
authors.lastname: Trotman
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Justin B
authors.initials: JB
authors.lastname: Kinney
authors.affiliation: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Sordella
mesh_terms: D000072283:A549 Cells; D000818:Animals; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D006801:Humans; D019426:Integrases / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism; D008024:Ligands; D051379:Mice; D000072417:Protein Domains; D055550:Protein Stability; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology; D013997:Time Factors
chemical_list: D064130:CRISPR-Associated Proteins; D008024:Ligands; D000094704:RNA, Guide, CRISPR-Cas Systems; D013629:Tamoxifen; C045073:Cre recombinase; D019426:Integrases