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

Author

Listed:
  • Krzysztof Chylinski

    (Vienna Biocenter (VBC))

  • Maria Hubmann

    (Vienna Biocenter (VBC))

  • Ruth E. Hanna

    (Broad Institute of MIT and Harvard)

  • Connor Yanchus

    (Mount Sinai Hospital
    University of Toronto)

  • 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
    University of Toronto)

  • Ulrich Elling

    (Vienna Biocenter (VBC))

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.

Suggested Citation

  • Krzysztof Chylinski & Maria Hubmann & Ruth E. Hanna & Connor Yanchus & Georg Michlits & Esther C. H. Uijttewaal & John Doench & Daniel Schramek & Ulrich Elling, 2019. "CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-13403-y
    DOI: 10.1038/s41467-019-13403-y
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