Abstract
The RNA-guided endonuclease Cas9, which
is involved in the prokaryotic CRISPR-Cas adoptive
immune system, binds to a guide RNA and cleaves
double-stranded DNA complementary to the RNA guide.
In recent years, Cas9 has been used as a versatile
genome-editing tool in a wide range of fields, from
fundamental research to clinical applications. However,
the molecular mechanism of DNA recognition and
cleavage by Cas9 was unknown, and many issues
remained to be addressed for its applications to genome
editing. We elucidated the crystal structure of S.
pyogenes Cas9, which is most widely used for genome
editing, in complex with the guide RNA and its target
DNA, thus providing the first insights into the
Cas9-mediated DNA cleavage mechanism. Furthermore,
we solved the crystal structures of Cas9 nucleases from
three different bacteria and those of Cas12a (Cpf1)
nucleases, which are also harnessed for genome editing.
Collectively, these structural studies illuminated the
mechanistic convergence and divergence in the
CRISPR-Cas nucleases, and paved the way for the
engineering of new genome-editing tools with improved
functionalities.
