Guide RNA functional modules direct Cas9 activity and orthogonality.

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 Cell

journal_title

Molecular cell

authors

Briner AE,Donohoue PD,Gomaa AA,Selle K,Slorach EM,Nye CH,Haurwitz RE,Beisel CL,May AP,Barrangou R

doi

10.1016/j.molcel.2014.09.019

subject

Has Abstract

pub_date

2014-10-23 00:00:00

pages

333-339

issue

2

eissn

1097-2765

issn

1097-4164

pii

S1097-2765(14)00751-5

journal_volume

56

pub_type

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