image: Using CRISPR/Cas9 nickase improves the accuracy of edits and increases the editable regions on the yeast genome. view more
Credit: Kyoto University
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."
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The paper "Precise genome-wide base editing by the CRISPR Nickase system in yeast" appeared 18 May 2017 in the Scientific Reports, with doi: 10.1038/s41598-017-02013-7
Kyoto University is one of Japan and Asia's premier research institutions, founded in 1897 and responsible for producing numerous Nobel laureates and winners of other prestigious international prizes. A broad curriculum across the arts and sciences at both undergraduate and graduate levels is complemented by numerous research centers, as well as facilities and offices around Japan and the world. For more information please see: http://www.kyoto-u.ac.jp/en
Journal
Scientific Reports