AbstractEndonuclease system CRISPR‐Cas9 represents a powerful toolbox for the budding yeast's Saccharomyces cerevisiae genome perturbation. The resulting double‐strand breaks are preferentially repaired via highly efficient homologous recombination, which subsequently leads to marker‐free genome editing. The goal of this study was to evaluate precise targeting of multiple loci simultaneously. To construct an array of independently expressing guide RNAs (gRNAs), the genes encoding them were assembled through a BioBrick construction procedure. We designed a multiplex CRISPR‐Cas9 system for targeting 6 marker genes, whereby the gRNA array was expressed from a single plasmid. To evaluate the performance of the gRNA array, the activity of the designed system was assessed by the success rate of the introduction of perturbations within the target loci: successful gRNA expression, followed by target DNA double‐strand breaks formation and their repair by homologous recombination led to premature termination of the coding sequence of the marker genes, resulting in the prevention of growth of the transformants on the corresponding selection media. In conclusion, we successfully introduced up to five simultaneous perturbations within single cells of yeast S. cerevisiae using the multiplex CRISPR‐Cas9 system. While this has been done before, we here present an alternative sequential BioBrick assembly with the capability to accommodate many highly similar gRNA‐expression cassettes, and an exhaustive evaluation of their performance.