Fig. 2

Markerless CRISPR-Cas9-mediated multigene integration in K. marxianus CBS 6556. a Schematic representation of the three-primer colony PCR test and example gels for single and multiple gene integrations into the ABZ1 locus. The length of the up- and down-stream homology regions is 700 bp. b Integration efficiency of single (EGFP), dual (EGFP, DSRED), and triple (EGFP, YFP, and DSRED) genes. c Relative fluorescence intensity of EGFP expressed from different loci as measured by fluorescence microplate reader. d Normalized expression level of fluorescent proteins when produced individually or simultaneously with one or two additional fluorescent proteins. All experiments were performed in biological triplicate. Bars represent the mean, while error bars represent the standard deviation