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Precise genome-wide base editing by the CRISPR Nickase system in yeast
Atsushi Satomura1,2, Ryosuke Nishioka1, Hitoshi Mori1
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Scientific Reports
|May 20, 2017
Summary
We developed a CRISPR Nickase system for precise genome editing, overcoming limitations of the CRISPR/Cas9 system. This new technology enables rapid, site-specific base editing in yeast with no off-target effects.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 is a powerful tool for genome editing in eukaryotes.
- CRISPR/Cas9 editing is limited to specific DNA sequences near the PAM site and guide RNA target.
- Existing CRISPR systems can exhibit off-target mutations.
Purpose of the Study:
- To develop a novel genome-wide base editing technology.
- To overcome the limitations of editable base ranges and off-target effects in CRISPR/Cas9 systems.
- To enable rapid and precise base editing in yeast.
Main Methods:
- Development of a CRISPR Nickase system utilizing a single Cas9 nickase.
- Application of the CRISPR Nickase system for genome-wide base editing.
- Integration of the CRISPR Nickase system with yeast gap repair cloning for mutant construction.
Main Results:
- The CRISPR Nickase system precisely edited bases up to 53 bp from the nicking site, expanding editable ranges.
- The system demonstrated no off-target editing, unlike the standard CRISPR/Cas9 system.
- Yeast mutants were constructed rapidly, within five days, using the combined CRISPR Nickase and gap repair cloning approach.
Conclusions:
- The CRISPR Nickase system offers a versatile and powerful technology for precise base editing.
- This system overcomes limitations associated with editable base ranges and off-target effects.
- It facilitates rapid, site-specific genome engineering in yeast.
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