Molecular Mechanism of CRISPR-Cas and Development of Novel Genome-Editing Tools Towards Gene Therapy
Osamu Nureki, Professor, The University of Tokyo
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). We have solved the crystal structures of Cas proteins, from 7 species, 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 Cas nucleases, 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 for genome editing.
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