A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization.

Šafarič Tepeš, Polona, Sordella, Raffaella (September 2021) A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization. Journal of Visualized Experiments (175). ISSN 1940-087X

URL: https://www.ncbi.nlm.nih.gov/pubmed/34542533
DOI: 10.3791/60685


The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

Item Type: Paper
Subjects: Investigative techniques and equipment > CRISPR-Cas9
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function
CSHL Authors:
Communities: CSHL labs > Sordella lab
SWORD Depositor: CSHL Elements
Depositing User: CSHL Elements
Date: 2 September 2021
Date Deposited: 23 Sep 2021 13:54
Last Modified: 23 Sep 2021 13:54
URI: https://repository.cshl.edu/id/eprint/40356

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