FBS Colloquia No.219Laboratory of Intracellular Membrane Dynamics

Molecular mechanism of CRISPR-Cas9 and structure-guided development of genome-editing tool / Molecular mechanism of physical channels

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide (non-coding) RNAs (sgRNAs). We have solved the crystal structures of Cas9, from 5 sources (984 a.a. to 1,629 a.a.), complexed with sgRNA and its target DNA at atomic resolutions. These high-resolution structures combined with functional analyses revealed the generality and diversity of molecular mechanism of RNA-guided DNA targeting by Cas9, and uncovered the distinct mechanisms of PAM recognition. On the basis of the structures, we succeeded in changing the specificity of PAM recognition, which paves the way for rational design of new, versatile genome-editing technologies. We have generated single guanine recognizing Cas9 variants and mostly PMA-less Cas9 variants, which would be very useful for base editing and gene modulation using dCas9 as well as widening target space. Recent progress in cryo-EM single particle analysis enables us to solve large membrane protein structures rapidly. I would like to present here new structures of two physical stimuli-sensing channels from human to uncover their molecular mechanisms