Abstract
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.
Highlights
CRISPR-Cas[9] is an efficient and versatile tool for genome engineering in many species
Induction of editing by modulating single guide RNAs (sgRNA) activity is a viable approach for studying phenotypes in temporal or special manner, and of essential genes, as it decouples selection for sgRNAexpressing cells from onset of editing
Several inducible editing systems have been reported till date, controlling activity either on the side of Cas[9] or sgRNA
Summary
CRISPR-Cas[9] is an efficient and versatile tool for genome engineering in many species. 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. CRISPR-Cas[9] systems provide highly efficient genome editing tools using engineered single guide RNAs (sgRNA) and a simple and robust RNA-DNA hybridization-based target recognition[1,2,3,4]. Methods for regulating sgRNA expression are mostly based on DOX-inducible Pol III transcription of sgRNAs, which suffer from leakiness or reduced editing efficiency[18,19,20]. None of these systems provides a method for sequential editing of two loci. We present a two-step strictly ordered editing methodology, allowing sequential induction of mutagenic events at two loci, and use it to analyze the order of mutations that trigger tumorigenesis in vivo
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