Yeast artificial chromosomes were originally synthesised to insert large fragments for the preparation of genomic libraries. They played a crucial role in the Human Genome Project, but in recent years the interest within the scientific community has decreased. Alas, with the advancements in molecular biology techniques, they could become a platform of interest for insertion, modification and expression of recombinant genetic elements in yeast cells. In this work, we prepared a modified yeast artificial chromosome based on the pYAC4 vector, where we replaced the telomeric regions to allow it to be amplified in multiple parts using the PCR method and replaced the selection markers for easier use in the laboratory strains of yeast Saccharomyces cerevisiae. We added cassettes to this modified yeast artificial chromosome for phenotypic monitoring of the inserts and compared the efficiency of the backbone and cassette assembly between the Gibson assembly method and homologous recombination in the yeast cells. The inserted cassettes were then modified in vivo using the CRISPR-Cas9 method directly on the artificial chromosome. The efficiency of cassette modification was evaluated phenotypically, we examined the impact of chromosome linearization on the growth of transformants and whether storage including freezing negatively affects the preservation of artificial chromosomes in yeast. We found out that homologous recombination in yeast cells is more time-consuming and labor-intensive than Gibson assembly but yields better results when assembling multiple and more complex fragments. The cassettes in the yeast artificial chromosome can be modified using the CRISPR-Cas9 system directly on the chromosome, and linearization of artificial chromosomes and storage of yeast cells at -80 °C do not have a negative impact on the yeast cell growth.
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