CRISPR-Switch Regulates SgRNA Activity by Cre Recombination for Sequential Editing of Two Loci

Overview

Journal Nat Commun

Specialty Biology

Date 2019 Dec 1

PMID 31784531

Citations 19

Authors

Krzysztof Chylinski

Maria Hubmann

Ruth E Hanna

Connor Yanchus

Georg Michlits

Esther C H Uijttewaal

John Doench

Daniel Schramek

Ulrich Elling

Affiliations

Soon will be listed here.

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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