Fig. 1
From: Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts
Schematic overview of the CRISPR–Cas9-assisted genome editing in O. polymorpha. a The Cas9 delivery vector pWYE3208. The expression of Cas9 was driven by the promoter PScTEF1. The OpMET2 5′ and OpMET2 3′ represent the ~ 1.5 kb of up- and downstream homologous arms of the gene OpMET2, respectively. zeoR represents the zeocin resistance gene. The plasmid pWYEN is the gRNA delivery vector. The gRNA was driven by the promoter PScSNR52 and ended with the terminator SC SUP4t . OpADE2 5′ and OpADE2 3′ represent the ~ 1.5 kb up- and downstream homologous arms of the gene OpADE2, respectively. G418R represents the G418 resistance gene. b Schematic illustration of markerless genome editing. (i) Integration of Cas9. (ii) Gene modification. (iii) Eviction of the gRNA delivery vector and restoration of the OpADE2 gene. (iv) Eviction of the Cas9 delivery vector and restoration of the OpMET2 gene. PAM is the abbreviation of protospacer adjacent motif. ET represents the editing template (s)