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  • Title: CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci.
    Author: Chylinski K, Hubmann M, Hanna RE, Yanchus C, Michlits G, Uijttewaal ECH, Doench J, Schramek D, Elling U.
    Journal: Nat Commun; 2019 Nov 29; 10(1):5454. PubMed ID: 31784531.
    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.
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