Fig. 2

Schematic of the CATS method. CRISPR-Cas9 cleavage induces homologous repair. An SNR52 promoter-driven RNA guide and a GAL-L promoter-driven CAS9 sequence are contained in a single endonuclease plasmid conferring NAT resistance. A template plasmid, with a URA3 marker, contains a sequence encoding a new tag and promoter-driven marker flanked by homology to the 3′ and 5′ ends of the GFP ORF. This template plasmid contains at either end a protospacer and corresponding PAM sequence, matching that cleaved by the expressed endonuclease. Upon galactose induction, both the genomic GFP ORF and the two sites in the template plasmid will be cleaved by the Cas9 endonuclease as indicated with the scissor icon. DSB-induced repair then can replace the GFP tag with the new template sequence