Open AccessCRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci
Author(s) -
Krzysztof Chylinski,
Maria Hubmann,
Ruth E. Hanna,
Connor Yanchus,
Georg Michlits,
Esther C. H. Uijttewaal,
John G. Doench,
Daniel Schramek,
Ulrich Elling
Publication year - 2019
Publication title -
nature communications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.559
H-Index - 365
ISSN - 2041-1723
DOI - 10.1038/s41467-019-13403-y
Subject(s) - crispr , genome editing , cas9 , subgenomic mrna , guide rna , biology , computational biology , homologous recombination , genetics , gene
CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.