RRC ID 58479
Author Huynh N, Wang S, King-Jones K.
Title Spatial and temporal control of gene manipulation in Drosophila via drug-activated Cas9 nucleases.
Journal Insect Biochem Mol Biol
Abstract Advances in CRISPR/Cas9 have revolutionized molecular biology and greatly facilitated the ability to manipulate gene function through the creation of precisely engineered mutants. We recently reported a collection of modular gateway-compatible Cas9/gRNA Drosophila lines to interfere with gene expression in a tissue-specific manner, including polytene tissues. However, most current in vivo CRISPR/Cas9 tools cannot temporally control the induction of Cas9 or gRNAs via external stimuli such as RU486. A drug-inducible CRISPR/Cas9 system would allow studying genes at later stages where early lethality is an issue. This would be especially useful when combined with tissue-specific expression of Cas9 or gRNAs, allowing for full spatiotemporal control. Here, we present a RU486-inducible version of Cas9 and also show that a Rapamycin-inducible Cas9, previously used in mammalian cell culture, works in Drosophila as well. Both RU486 and rapamycin-inducible Cas9 work in vivo and in Drosophila cell culture. We also present split Cas9 constructs for rapamycin-dependent gene disruption and activation. These approaches establish drug-inducible and thus temporally controlled CRISPR/Cas9 tools for gene disruption and expression in a living model organism. Our CRISPR/Cas9 vector collection can be easily adapted for any tissue and provides higher fidelity compared to RNAi approaches.
Volume 120
Pages 103336
Published 2020-5-1
DOI 10.1016/j.ibmb.2020.103336
PII S0965-1748(20)30025-4
PMID 32105778
MeSH Animals Base Sequence CRISPR-Cas Systems* Drosophila / genetics* Endonucleases Gene Editing* Gene Expression*
IF 3.827
Times Cited 0