122nd General Meeting of the KCS

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