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Fig. 1 | Genome Biology

Fig. 1

From: Pop in, pop out: a novel gene-targeting strategy for use with CRISPR-Cas9

Fig. 1

Two step, ‘pop in & out’ approach for the generation of CRISPR-Cas9-induced targeted mutants. a In the ‘in’ step, the targeting vector (homology regions shaded gray) introduces a tag segment that is disrupted by a loxP-flanked green fluorescent protein (GFP) reporter gene. A Cas9 and sgRNA-induced double strand break (DSB) stimulates homology-directed repair (HDR) and enables the enrichment of targeted GFP+ cells by fluorescence-activated cell sorting (FACS). In the ‘out’ step, the marker is deleted by Cre/lox-mediated recombination and GFP cells are subsequently enriched by FACS. b Two step in & out targeting approach for the seamless removal of a marker gene. In the first step, the targeting vector introduces a nucleotide replacement (a single nucleotide polymorphism (SNP)) next to a GFP reporter. The marker is removed from the targeted allele using Cas9 and a pair of sgRNAs that recognize the end of the marker cassette. The marker gene is removed by HDR with a targeting vector to provide sequences (homology regions shaded) that are wild type except for the SNP, and GFP cells are subsequently enriched by FACS

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