Fig. 8

Simultaneous conversion to dual targeting constructs using two endonucleases. a Schematic of the targeting process with two different DNA templates, to replace two regions of the genome. The endonuclease plasmid contains a GAL-L promoter-driven CAS9 gene and two SNR52 promoter-driven sgRNA sequences, one targeting GFP and the other CAN1. The dual-template plasmid contains two template sequences, one to replace CAN1 with HYG, and the other to replace GFP with an RFP-ADH1 promoter-Kanamycin sequence, as demonstrated in Fig. 2. Each is flanked by homology to the endogenous gene, and by the relevant protospacers, so that each endonuclease complex will cut both the genome and the plasmid template sequence, inducing HDR into both loci. b Proportions of converted strains from the dual targeting experiment illustrated in (a). CAN1 targeting confers HYG resistance, and conversion from GFP to RFP confers G418 resistance. The strain Htb2-GFP contained the dual-template plasmid alongside an endonuclease plasmid encoding both the GFP and CAN1 guides, or each of these individually, as indicated. The proportions are calculated against the number of colonies growing on YPD without selection, and shown here is the mean and standard deviation (error bars) of 3 biological replicates