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

Genomic engineering methods represent powerful tools to examine chromosomal modifications and to subsequently study their impacts on cellular phenotypes. However, quantifying the fitness impact of translocations, independently from base substitutions or the insertion of genetic markers, remains a challenge. Here we report a rapid and straightforward protocol for engineering either targeted reciprocal translocations at the base pair level of resolution between two chromosomes or multiple simultaneous rearrangements in the yeast genome, without inserting any marker sequence in the chromosomes. Our CRISPR/Cas9-based method consists of inducing either (1) two double-strand breaks (DSBs) in two different chromosomes with two distinct guide RNAs (gRNAs) while providing specifically designed homologous donor DNA forcing the trans-repair of chromosomal extremities to generate a targeted reciprocal translocation or (2) multiple DSBs with a single gRNA targeting dispersed repeated sequences and leaving endogenous uncut copies of the repeat to be used as donor DNA, thereby generating multiple translocations, often associated with large segmental duplications (Fleiss, et al. PLoS Genet 15:e1008332, 2019).