Fig. 1

DNA-free RNP-mediated GE strategy applied to C. r.CW15. (A) CW15 synchronized cells were transformed by electroporation with RNPs (assembled with Cas9, T1, and T2) to target the sequence of CpFTSY. (B) Transformed cells were plated on TAP agar plates, and potential KO cells were selected based on their pale-green phenotype. Then, colonies were streaked on new TAP agar plates to obtain individual and clean mutant lines. (C) Schematic CRISPR sgRNA design for knocking-out CpFTSY gene PAM sequences are shown. T1 and T2 are in exons 4 and 6, at position 1718 bp and 2656 bp, respectively (Fig. 1S, Table 1S). (D) Individual mutants were grown in liquid culture; PCR and Sanger sequencing were carried out after DNA extraction to pinpoint Cas9-induced mutations. (E) Sequencing data output of 17 pale-green colonies, showing the two target regions T1 and T2. Wild type sequences are highlighted in blue, Cas9 specific PAM motif in yellow. Missing nucleotides are indicated as “-”, nucleotide substitutions are highlighted in green