CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

Krzysztof Chylinski, Maria Hubmann, Ruth E. Hanna, Connor Yanchus, Georg Michlits, Esther C. H. Uijttewaal, John Doench, Daniel Schramek and Ulrich Elling ()
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Krzysztof Chylinski: Vienna Biocenter (VBC)
Maria Hubmann: Vienna Biocenter (VBC)
Ruth E. Hanna: Broad Institute of MIT and Harvard
Connor Yanchus: Mount Sinai Hospital
Georg Michlits: Vienna Biocenter (VBC)
Esther C. H. Uijttewaal: Vienna Biocenter (VBC)
John Doench: Broad Institute of MIT and Harvard
Daniel Schramek: Mount Sinai Hospital
Ulrich Elling: Vienna Biocenter (VBC)

Nature Communications, 2019, vol. 10, issue 1, 1-12

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-13403-y

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