A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9.

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Genomic engineering tools are vital for studying chromosomal modifications and their cellular effects.
• Quantifying the fitness impact of chromosomal translocations, separate from other genetic changes, is difficult.

Purpose of the Study:

• To develop a rapid and precise method for engineering chromosomal translocations in yeast.
• To enable the study of translocation fitness impacts independently of other genetic alterations.

Main Methods:

• Utilizing CRISPR/Cas9 technology to induce targeted double-strand breaks (DSBs) in yeast chromosomes.
• Employing specifically designed homologous donor DNA or endogenous repeats to facilitate trans-repair and generate translocations.
• Engineering single reciprocal translocations or multiple simultaneous rearrangements without marker insertion.

Main Results:

• A straightforward protocol for base-pair level resolution of reciprocal translocations between chromosomes.
• The ability to generate multiple simultaneous translocations, often accompanied by segmental duplications, using a single guide RNA targeting dispersed repeats.
• Successful genomic engineering in yeast without the need for inserting genetic marker sequences.

Conclusions:

• The developed CRISPR/Cas9-based method offers a powerful and efficient approach for precise genomic engineering of chromosomal translocations in yeast.
• This technique overcomes previous challenges in quantifying translocation fitness impacts by excluding confounding factors like base substitutions or marker insertions.
• The protocol facilitates advanced research into chromosomal instability and its functional consequences in cellular phenotypes.