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.

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-ER, 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.