Overcoming the Limitations of CRISPR-Cas9 Systems in : Off-Target Effects, Epigenome, and Mitochondrial Editing.

Modification of the genome of the yeast has great potential for application in biological research and biotechnological advancements, and the CRISPR-Cas9 system has been increasingly employed for these purposes. The CRISPR-Cas9 system enables the precise and simultaneous modification of any genomic region of the yeast to a desired sequence by altering only a 20-nucleotide sequence within the guide RNA expression constructs. However, the conventional CRISPR-Cas9 system has several limitations. In this review, we describe the methods that were developed to overcome these limitations using yeast cells. We focus on three types of developments: reducing the frequency of unintended editing to both non-target and target sequences in the genome, inducing desired changes in the epigenetic state of the target region, and challenging the expansion of the CRISPR-Cas9 system to edit genomes within intracellular organelles such as mitochondria. These developments using yeast cells to overcome the limitations of the CRISPR-Cas9 system are a key factor driving the advancement of the field of genome editing.