Fig. 2

DNA-free GE strategy co-targeting cpftsy and psr1. (A) CW15 synchronized cells were transformed by electroporation with RNPs of sgRNA guided to both the target sequences T1 and T2 of CpFTSY and target sequences T1 and T2 of PSR1. T1 and T2 of PSR1 were chosen in exon 1 and 3, at position 295 bp and 1768 bp, respectively (Fig. 8S). (B) A first selection round was performed on TAP agar plates based on a pale-green phenotype. Selected colonies were streaked on new TAP agar plates to permit the isolation of single, genetically identical colonies. (C) A second screening was performed on TA agar plates devoid of phosphate and supplemented with BCIP, the latter used for the colorimetric detection of alkaline phosphatase activity. Colonies expressing PSR1 exhibited both blue and halo, while colonies devoid of functional PSR1 cannot process BCIP by PSR1-regulated phosphatases and resulted green. (D) Potential double mutants, highlighted with yellow arrows, were analyzed by Sanger sequencing of target regions of both CpFTSY (Fig. 1C) and PSR1 (E). (F) Sequencing data output of PSR1 and CpFTSY target regions T1 and T2, wild type sequences are highlighted in blue, and specific PAM motifs are in yellow. Missing nucleotides are indicated as “-” and nucleotide substitutions are highlighted in green. BCIP = 5-bromo-4-chloro-3-indolyl phosphate