Crystal structure of the CRISPR RNA–guided surveillance complex from Escherichia coli

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405 kDa multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 Å resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a sea-horse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3′- and 5′-ends of the crRNA are anchored by proteins at opposite ends of the complex, while the guide sequence is displayed along a helical assembly of six interwoven subunits that present 5-nucleotide segments of the crRNA in pseudo A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.

Rational design of a split-Cas9 enzyme complex

“…Our results also invite comparisons to the mechanisms of RNP assembly and DNA targeting in other CRISPR-Cas systems, particularly the type I-E Cascade interference complex. Although Cascade is composed of 11 distinct subunits, none of which possesses nuclease activity, the guide RNA (crRNA) plays a similarly critical role in scaffolding the assembly of distinct domains into a structure that is primed to engage DNA targets (19)(20)(21). Similar principles are likely to govern the assembly and activity of other CRISPR RNA-guided DNA-targeting complexes (2).…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…For both the Cas9 and the class 1 Cascade complex, a remarkable feature of the transition from the pre-target-bound binary state to the target-bound ternary state involves the formation of a preordered A-form crR-NA, either only within the seed region (Cas9) [14] or throughout the entire guide region (Cascade complex) [31][32][33]. This strategy is also employed by the eukaryotic Agronaute complexes during the transition from guide-RNA bound form to target transcript recognition [28][29][30], representing a convergent evolution of this mechanism [14].…”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…In vivo, several copies of Cas7 proteins are wrapped around crRNA in a sequence-unspecific helical fashion [5,30,50,51]. Crystal structures from single and complex-bound Cas7 proteins show two composite RNA-binding surfaces: a central cleft and a structurally variable insertion domain [5,23,24,52]. In all Cas7 proteins characterized to date both these domains are defined by insertions within the secondary structure elements of the central RRM domain (insertion domain 1 is b1-a1, b2-b3, a2-b4, and insertion domain 2 is a1-b2).…”

“…We compared our results with the crRNA-binding surface of Type I-E Escherichia coli Cas7, which was crystallized in context of the fully assembled crRNP complex from E. coli [5]. For this, the crystal structure of T. pendens Cas7 and the homology models (T. tenax Cas7 and T. thermophilus Csm3) were superposed onto two copies of E. coli Cas7 (PDB ID: 1VY8) using secondary-structure matching (SSM) superposition in COOT [54].…”

“…The ''gold standard'' for characterizing molecular interactions of RBDs with their cognate RNA molecules by structure determination is co-crystallization [4,5]; others include NMR of the complex [6], or high-resolution EM of entire RNPs, as performed for the ribosome [7]. Although the number of co-structures of RBPs has been steadily increasing with more than 200 co-structures of protein-RNA complexes available in the PDB, most RBPs are still crystallized without RNA.…”