Nicking in new nucleotides

Kyoto University CRISPR/Cas9 'nickase' system a powerful new tool in yeast genomics

Kyoto, Japan -- Who in genomics hasn't heard about CRISPR/Cas9?

In the five years since the gene-targeting and editing tool was developed, it has effectively revolutionized the field of biology with its cut-and-paste simplicity.

But CRISPR has its problems: the efficiency of gene editing remains relatively low due to a high rate of inaccurate edits.

In order to improve CRISPR's applicability, a Kyoto University team has used yeast to develop a genome-wide base-editing technology which they have successfully shown to improve editing accuracy. Their research on this new "CRISPR Nickase system" appeared recently in Scientific Reports .

The new system relies on two components, a guide RNA -- 'gRNA' -- and the Cas9 nickase. The gRNA first targets a specific location of the genome, which the nickase then detects and cuts.

"When editing the yeast genome, we found frequent inaccuracies in DNA regions outside of the gRNA target," explains Atsushi Satomura, first author of the study. "As a workaround, we employed the Cas9 nickase, a variant of Cas9 which only cuts one strand of the DNA double helix."

By "nicking" the DNA instead of fully cutting both strands, the team found that the rate of inaccurate mutations was dramatically reduced.

"We applied this system with a method called 'yeast gap repair cloning', or GRC, to streamline the generation of yeast mutants," continues Satomura. "This is considerably faster than before. Previous methods required a complex procedure that took nearly two weeks."

Combining the nickase system and GRC shortened that time to five days.

"In theory, we can now accurately edit 97.2% of the bases in the yeast genome," concludes team leader Mitsuyoshi Ueda. "We hope our new system will be a robust tool that overcomes CRISPR's limitations, not only for yeast genomics but in other animal systems as well."