Figure 5.

Cas9-based marker-less, scar-less genomic integration. (A) Shows workflow, where first a marker-less integration cassette with flanking regions of 500 bp of homology to the chromosome and the RSF1010-Cas9-gRNA plasmid are co-transformed into a parent strain. If co-transformation is successful, the native homologous recombination machinery of ADP1 will exchange alleles, and Cas9 will be unable to cut the target locus, allowing survival for the RSF1010 plasmid. Following, streaking on non-selective medium clears the plasmid. (B) Shows fluorescent expression from marker-less integration of lacI-Trc-mCherry at both the vanAB and pcaHG loci. Error bars are standard deviation for biological triplicate.