|
Type |
Oral Presentation |
Area |
Oral Presentation of Young Biological Chemists |
Room No. |
Room 314 |
Time |
THU 10:15-: |
Code |
LIFE.O-5 |
Subject |
Target specificity of the CRISPR-Cas9 nuclease regulated by the REC2 domain via structural rearrangement of DNA |
Authors |
Keewon Sung, Jinho Park, Nam Ki Lee, Seong Keun Kim* Department of Chemistry, Seoul National University, Korea |
Abstract |
The RNA-guided CRISPR-Cas9 nuclease cleaves DNA targets using recognition by base-pairing between the RNA and DNA sequences. Originating from an adaptive immune system in prokaryotes, CRISPR-Cas9 has been widely studied for genome engineering applications, but the mechanistic basis of its target specificity still remains to be elucidated to understand and minimize off-target DNA cleavage. In this work, using single-molecule FRET and REC2-mutational analyses, we show that a non-nucleolytic domain within the Cas9 nuclease called REC2 plays a crucial role in enhanced off-target discrimination. Specifically, we find that the REC2 domain regulates structural rearrangement of the non-target strand of DNA for cleavage reaction with the help of positively-charged amino acids on its surface, by inducing a non-cleavable conformation particularly for off-target sequences. This result provides molecular insights for the target-specific activation of the Cas9 nuclease and thereby for rational design of engineered Cas9 toward highly-specific genome editing. |
E-mail |
kwsung@mrd.snu.ac.kr |
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