Structural basis for CRISPR RNA-guided DNA recognition by Cascade

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

“…S6B). These results may suggest that stable docking of Cas3 and Cse1 requires other components of Cascade complex, perhaps to induce the conformational change that is observed in Cse1 within Cascade when DNA is bound (13,43). It is also possible that a defined R-loop structure needs to be present to facilitate correct orientation of Cascade to allow Cas3-Cse1 interaction or other Cascade subunits are partly involved in the formation of a stable complex with Cas3.…”

“…Whereas Cas9 and Csm/Cmr effector complexes directly recognize and nucleolytically degrade invading genes (12,14,16,17,21), Cascade complex by itself is not a nuclease for degradation of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13).…”

“…Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7,8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9)(10)(11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12)(13)(14)(15)(16)(17)(18).…”

“…In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13). Formation of the R-loop structure induces conformational change in Cascade subunits, triggering recruitment of Cas3 protein that is a nuclease-helicase responsible for degradation of dsDNA (27)(28)(29)(30).…”

“…Cascade (Cas complex for antiviral defense) complexes are synonymous with interference in type I CRISPRs, comprising multiple proteins in a helical structure around crRNA that targets invader DNA (13,19,20). Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12,(21)(22)(23)(24).…”