Sternber, S.H. et al. Nature (2015) 527:110-113. http://www.ncbi.nlm.nih.gov/pubmed/26524520
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.