Fig. 1
From: CRISPR-FRT targets shared sites in a knock-out collection for off-the-shelf genome editing
Overview of the CRISPR-FRT protocol. CRISPR-FRT makes use of the arrayed collection of Keio knockout mutants having a FRT-flanked kanamycin-resistance (KanR) cassette replacing each non-essential E. coli gene. CRISPR-FRT includes a gRNA-FRT that directs the Cas9 nuclease to bind and cut the two FRT sites. A convenient rescue template (e.g., a mutated gene from an evolved E. coli strain amplified by PCR, a plasmid-encoded gene variant, etc) recombines (dashed lines) over the homologous regions flanking the KanR cassette. Survivors are screened to separate KanR false positives (red X) from the kanamycin sensitive (KanS) true positives (green check) that replaced KanR cassette with the mutated gene
