Conformational control of DNA target cleavage by CRISPR–Cas9

Sternber, S.H. et al. Nature (2015) 527:110-113.

One of the major obstacles facing CRISPR/Cas9 systems is off-target cleavage events.  Understanding the mechanisms by which CRISPR/Cas9 systems identify and cleave DNA could provide insights into ways to decrease off-target cleavage.  While Cas9 crystal structures have been elucidated the HNH domain active site was found to be ~30Å away from the targeted DNA thus preventing a detailed understanding of the cleavage mechanism.  By developing a Förster resonance energy transfer (FRET) based system Sternber et al were able to determine that two α-helices induce a conformational shift that acts as a signal transducer to activate both the HNH and RuvC nuclease domains thus coordinating cleavage of both DNA strands.  Furthermore, the authors determined that Cas9 could cleave off-target sites that contained only 1-3 mismatches a the distal end of the guide RNA since the activating conformation shift was still able to occur.

Author: Advanced Analytical

Advanced Analytical Technologies, Inc. (AATI) simplifies complex genomics workflows to accelerate research and discovery in pharmaceuticals, life science, biofuels, biotechnology and healthcare.

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