Abstract:
:The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.
journal_name
Mol Celljournal_title
Molecular cellauthors
Briner AE,Donohoue PD,Gomaa AA,Selle K,Slorach EM,Nye CH,Haurwitz RE,Beisel CL,May AP,Barrangou Rdoi
10.1016/j.molcel.2014.09.019subject
Has Abstractpub_date
2014-10-23 00:00:00pages
333-339issue
2eissn
1097-2765issn
1097-4164pii
S1097-2765(14)00751-5journal_volume
56pub_type
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