Precise genome-wide base editing by the CRISPR Nickase system in yeast

TLDR

The CRISPR/Cas9 system has enabled efficient genome editing in many eukaryotic cells, but its editable bases are limited to those within the protospacer adjacent motif (PAM) and guide RNA‑targeting sequences. This study developed a genome‑wide base‑editing technology, the CRISPR Nickase system, that uses a single Cas9 nickase. By coupling the CRISPR Nickase system with yeast gap‑repair cloning, the authors constructed yeast mutants in only five days. The system overcomes the PAM limitation, precisely edits bases up to 53 bp from the nicking site, shows no off‑target editing, and provides a versatile, rapid, site‑specific, and precise base‑editing platform in yeast.

Abstract

Abstract The CRISPR/Cas9 system has been applied to efficient genome editing in many eukaryotic cells. However, the bases that can be edited by this system have been limited to those within the protospacer adjacent motif (PAM) and guide RNA-targeting sequences. In this study, we developed a genome-wide base editing technology, “CRISPR Nickase system” that utilizes a single Cas9 nickase. This system was free from the limitation of editable bases that was observed in the CRISPR/Cas9 system, and was able to precisely edit bases up to 53 bp from the nicking site. In addition, this system showed no off-target editing, in contrast to the CRISPR/Cas9 system. Coupling the CRISPR Nickase system with yeast gap repair cloning enabled the construction of yeast mutants within only five days. The CRISPR Nickase system provides a versatile and powerful technology for rapid, site-specific, and precise base editing in yeast.