PMC

Diagram of Yeast Oligo-mediated Genome Engineering (YOGE) applications. Synthetically assembled foreign DNA or natural genomes can be modified with YOGE. Iterative rounds can be used to increase the frequency and/or diversity of the directed modifications. Finally, strains can be screened or selected to isolate desired genotypic or phenotypic traits.

Oligonucleotide recombination frequency across all strains and loci tested. Recombinations were completed using 90mer oligonucelotides with the optimal sense strand targeted. All recombinations were completed in triplicate, with values plotted to represent the mean values and error bars corresponding to the standard deviation. A. Oligonucleotide incorporation frequency in strain VL6-48 B. Oligonucleotide incorporation frequency in strain CEN.PK113-7D C. Oligonucleotide incorporation frequency in strain VTT C-68059a.

Cycling and multiplex modifications using YOGE in strain CEN.PK.RR at the CAN1 and LYP1 loci. All values represent the mean of three replicates with error bars representing the standard deviation. A. Diagram of YOGE electroporation cycling and recovery. B. Singleplex recombination frequencies for the CAN1 and LYP1 loci. C. Multiplex recombination frequency for both loci simultaneously. D. Rate of gene conversion without oligonucleotide added during electroporation, demonstrating that enrichment in loci modification is caused from transformation and not enrichment due to fitness advantage of any genotype.

Variability in oligonucleotide recombination across DNA strands, loci and strains. All values represent the mean of three replicates with error bars representing the standard deviation. A. Diagram of the transcription fork with the coding and noncoding strands indicated. Oligonucleotides are named based on the sequence they correspond to in the transcription fork. B. Strand bias variability in oligonucleotide recombination across the ADE2, LEU2, and CAN1 loci of the VL6-48.RR strain. C. Strand bias variability in oligonucleotide recombination across the URA3, CAN1, and LYP1 loci of the CEN.PK.RR strain D. Strand bias variability in oligonucleotide recombination across the URA3, CAN1, and LYP1 loci of the VTT.RR strain.

External factors affecting oligonucleotide recombination. All values represent mean values of three replicates with error bars representing the standard deviation. A. Effect of oligonucleotide length on recombination at the ADE2 locus in strain VL6-48.RR B. Effect of oligonucleotide concentration on recombination at the ADE2 locus in strain VL6-48.RR C. Effect of mismatches, deletions and insertions on the oligonucleotide recombination frequency at the CAN1 locus in strain VL6-48.RR (the 1 mismatch, all deletion, and all insertion experiments were completed with 5 replicates) D. The survival rate of cells following electroporation across the three strains tested. E. Frequency of plasmid transformation in all three strains using a centromeric plasmid containing hygromycin resistance.