Genome manipulation, especially the deletion or replacement of chromosomal regions, is a salient tool for the analysis of genome function. Because of low homologous recombination activity, however, current methods are limited to manipulating only one chromosomal region in a single transformation, making the simultaneous deletion or replacement of multiple chromosomal regions difficult, laborious, and time-consuming. Here, we have developed two highly efficient and versatile genome engineering technologies, named clustered regularly interspaced short palin-dromic repeats (CRISPR)-PCR-mediated chromosomal deletion (PCD) (CRISPR-PCD) and PCR-mediated chromosomal replacement (CRISPR-PCRep), that integrate the CRISPR-associated protein 9 (Cas9) genome editing system (CRISPR/Cas9) into, respectively, the PCD method for chromosomal deletion and our newly developed PCRep method for chromosomal replacement. Integration of CRISPR induces double strand breaks to activate homologous recombination, and thus enhances the efficiency of deletion by PCD and replacement by PCRep, enabling multiple chromosomal regions to be manipulated simultaneously for the first time. Our data show that CRISPR-PCD can delete two internal or terminal chromosomal regions, while CRISPR-PCRep can replace triple chromosomal regions simultaneously in a single transformation. Colony PCR analysis of structural alterations showed that triple replacement of four different sets of chromosomal regions was successful in 83%-100% of transformants analyzed. These novel genome engineering technologies, which greatly reduce time and labor for genome manipulation, will provide powerful tools to facilitate the simultaneous multiple deletion and replacement of chromosomal regions, enabling the rapid analysis of genome function and breeding of useful industrial yeast strains. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.