A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Traditional yeast genome modification relied on selectable markers, limiting gene editing capabilities.
• CRISPR-Cas9 technology enables targeted DNA modifications without permanent markers.

Purpose of the Study:

• To detail an improved method for incorporating guide RNA (gRNA) sequences into CRISPR-Cas9 expression plasmids for yeast genome editing.
• To demonstrate the application of this enhanced CRISPR-Cas9 protocol for introducing various genetic modifications in yeast.

Main Methods:

• Utilized Golden Gate cloning for efficient ligation of annealed oligonucleotides into directional restriction enzyme sites.
• Developed a streamlined CRISPR-Cas9 system for generating DNA double-strand breaks (DSBs) at specific genomic locations.
• Employed PCR-derived DNA repair templates for homologous recombination-mediated repair of DSBs.

Main Results:

• Successfully incorporated gRNA-encoding DNA sequences into the Cas9 expression plasmid using Golden Gate cloning.
• Demonstrated the efficacy of the improved CRISPR-Cas9 method for introducing multiple types of directed genetic changes in the budding yeast genome.
• The protocol facilitates precise genomic modifications without the need for permanent selectable markers.

Conclusions:

• The described method offers an efficient and versatile approach for CRISPR-Cas9 mediated genome editing in yeast.
• This improved protocol overcomes limitations of previous methods, enabling more flexible and precise genetic engineering in budding yeast.
• The findings contribute to advancing genome editing technologies in microbial systems.