SH3 Domain Chimeric Variant Construction

Chimeric variants of divergent substitutions between the two SH3 domains with single and double amino substitutions and the wildtype sequence with 20bp homology arms on either side were synthesized as oligonucleotide pools (Integrated DNA Technologies Ltd) for insertion at the native loci. The genomic insertion of the synthesized libraries was performed by targeting the stuffer sequence in the MYO3 and MYO5 SH3-deleted strains. MYO3 and MYO5 SH3-deleted yeast competent cells were co-transformed with pCAS (Addgene plasmid 60847) containing the stuffer-specific gRNA and Streptococcus pyogenes Cas9^{29 (link),45 ,50} with the PCR amplified MYO3 and MYO5 SH3 chimeric variant synthesized libraries acting as the donor DNA, respectively. We targeted ~1000 colonies on each plate, which were retrieved by washing with sterile YPD. Glycerol stocks of yeast cells were kept for each position (2 different stocks corresponding to the chimeric variants of Myo3 SH3 in MYO3 and Myo5 SH3 in MYO5). The stocks were validated using MiSeq Reagent Kit v3 on an Illumina MiSeq for 600 cycles for paired-end 300 sequencing (IBIS sequencing platform, Université Laval, Québec).