Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

Abstract

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. While Type I and Type II CRISPR-Cas surveillance complexes target double-stranded DNA, Type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy (cryo-EM) reconstructions of native Type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumb-like β-hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nt intervals. The Cmr complex is architecturally similar to the Type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.Bacteria and archaea defend themselves against infection using adaptive immune systems comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and CRISPR-associated (Cas) genes (1). A defining feature of CRISPR-Cas systems is the use of Cas proteins in complex with small CRISPR RNAs (crRNAs) to identify and cleave The effector complex of the Type III system from T. thermophilus (Cmr) is a 12-subunit assembly composed of six Cmr subunits (Cmr1-6) and a crRNA with a stoichiometry of Cmr1 1 2 1 3 1 4 4 5 3 6 1 :crRNA 1 (7). The Cmr complex binds to target RNA that is complementary to the bound 40 or 46-nt crRNA and cleaves the target at 6-nt intervals measured from the 5' end of the crRNA sequence (7,8). Although low-resolution structural studies revealed an overall capsule-like architecture of the Cmr complex (7), the molecular basis of subunit assembly, crRNA binding and ssRNA target recognition and cleavage by the intact surveillance complex remains unknown.We performed cryo-electron microscopy (cryo-EM) of the intact ~350-kDa Cmr complex in the absence and presence of target ssRNA. We purified endogenous apo-Cmr (containing a crRNA) and used this sample for step-wise assembly with a 56-nt biotinylated ssRNA target followed by purification using streptavidin affinity chromatography. Frozen-hydrated samples of both apo-Cmr and target-bound Cmr were visualized using an FEI Titan Krios microscope equipped with a Gatan K2 Summit direct electron detector. Cryo-EM micrographs of both apo-Cmr and the ssRNA-bound complex showed mono-disperse, easily identifiable particles with sea worm-like features ( fig. S1). Using LEGINON (9), we acquired ~7,000 and ~4,000 micrographs and automatically picked ~700,000 and ~300,000 apo-and target-bound Cmr particles, respectively, using Appion (10). After 3D classification and single-particle reconstruction (Supplementary Material and Methods) in RELION (11), we obtained structures of intact apo-Cmr and target-bound Cmr at ~4.1 and 4.4-Å resolution ( fig. S1, S2) (using the 0.143 gold standard Fourier Shell Correlationcalc...

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease

“…The interaction of the Cmr complex with target RNA is guided by crRNA/target RNA complementary base-pairing (Hale et al 2009(Hale et al , 2012Ramia et al 2014). Multiple Cmr4 subunits, which form the backbone of the complex, mediate cleavage of the bound target RNA at regular 6-nt intervals (Staals et al 2013;Benda et al 2014;Hale et al 2014;Ramia et al 2014;Taylor et al 2015). Recent data indicate that the Cmr system of Sulfolobus islandicus is capable of transcription-dependent, plasmid silencing in vivo, although this activity has not been recreated with purified components or characterized in detail (Deng et al 2013).…”

Cutting it Right: Plasticity and Strategy of CRISPR RNA Specific Nucleases

“…This allows sharing of crRNA processing pathways, with the mature crRNAs ultimately getting loaded onto specific effector complex. Type III system displays two kinds of effector complexes -Csm (Type III-A/D) or Cmr (Type III-B/C) (Hale et al, 2009;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Tamulaitis et al, 2014;Zhang et al, 2016), which show distant relationship with Type I effector complex (Rouillon et al, 2013;Osawa et al, 2015;Taylor et al, 2015).…”

“…However, in case of type III systems, pre-CRISPR transcript is processed by Cas6 in solitude and not as part of the effector complex (Carte et al, 2008; Carte et al, 2010;Wang et al, 2011;Shao and Li, 2013;Shao et al, 2016; Makarova et al, 2017a). Further, Cas6/III does not become a constituent of interference complex (Hale et al, 2009;Zhang et al, 2012;Rouillon et al, 2013;Spilman et al, 2013; Benda et al, 2014;Staals et al, 2014;Osawa et al, 2015;Taylor et al, 2015; Makarova et al., 2017a), with the exception being Csm/III-A complex from Streptococcus thermophilus that shows weak transient interactions with Cas6 (Tamulaitis et al, 2014). This possibly grants Cas6/ III the flexibility required to associate with multiple subtypes that potentially differ at the interference stage.…”

“…This allows sharing of crRNA processing pathways, with the mature crRNAs ultimately getting loaded onto specific effector complex. Type III system displays two kinds of effector complexes -Csm (Type III-A/D) or Cmr (Type III-B/C) (Hale et al, 2009;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Tamulaitis et al, 2014;Zhang et al, 2016), which show distant relationship with Type I effector complex (Rouillon et al, 2013;Osawa et al, 2015;Taylor et al, 2015).A primary processed crRNA typically contains 8 nt repeat sequence at the 5'-end (often called 5' handle), a spacer sequence (guide) (Brouns et al, 2008; Haurwitz et al, 2010; Lintner et al, 2011) and a variable 3'-end in some instances, which stems from further trimming at 3'-end by as yet unidentified nuclease(s) (Hale et al, 2008; Hatoum-Aslan et al, 2011;Richter et al, 2012;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Shao and Li, 2013;Tamulaitis et al, 2014). Intriguingly, the extent of processing appears to be effector complexspecific (Hale et al, 2009; Lintner et al, 2011;Zhang et al, 2012; Hatoum-Aslan et al, 2013;Rouillon et al, 2013;Tamulaitis et al, 2014).…”

Profile of Eva Nogales

“…For the last five years, we've been working on CRISPR/Cas9 complexes," says Nogales. Most recently, their studies revealed how thumb-like subunits of the CRISPR-Cmr complex extend into the target RNA and facilitate its cleavage (10). In 2012, Nogales and biochemist Andreas Martin, whom she had convinced of cryo-EM's applicability to his study of proteosomes, published the structure of more than 30 subunits of the yeast 26S proteasome, revealing the mechanistic underpinnings of ubiquitin-dependent protein degradation (11).…”