The CRISPR RNA-guided surveillance complex inEscherichia coliaccommodates extended RNA spacers

Abstract

Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli, CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.

Altered stoichiometryEscherichia coliCascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

“…In contrast, g8mut only slightly affected the binding kinetics, indicating that the competition effect caused by g8cons results from its specific binding to Cascade. Assuming that the beacon binding rate in the presence of g8cons is proportional to concentration of Cascade molecules unoccupied by g8cons, a Kd for g8cons binding to wt Cascade is 0.7 nM, consistent with earlier reported Kd values (18,32). Competition binding experiments with other Cascade-crRNA complexes (Supplementary Figure S4 and Table S4) showed that affinities of +6 Cascade and −3 Cascade to g8cons were similar to wild-type (Kd values ∼1 nM), whereas −6 Cascade and −12 Cascade formed less stable complexes (Kd values ∼15 nM).…”

“…Very recently, evidence has been presented that crRNAs from two different type I CRISPR-Cas systems with longer than normal spacers assemble into effector complexes containing larger than the normal complement of six Cas7 monomers and capable of CRISPR interference (18,19). Cascades with shortened crRNA spacers were reported to be inactive, however (19).…”

“…Cascade complex samples were sprayed from in-house prepared gold-coated borosilicate glass capillaries and analyzed on a SYNAPT G2-Si instrument (Waters) as reported before (18). Briefly, purified Cascade complexes were buffer exchanged to 100 mM ammonium acetate, pH 7 (Sigma) using 3 kDa molecular weight cutoff spin filters (Pall Corp.) and infused to electrospray source at protein concentration of 2–3 μM and the rate around 90 nl/min.…”

“…In fact the spacer length may dictate the oligomerization state of such subunits, leading to production of effector complexes with different subunit stoichiometries and functional properties. Indeed, a recent report showed that the E. coli crRNA spacer, normally 32-nt long, can be extended leading to formation of functional Cascade complexes with extra Cas7 monomers (18). Functional Cascades with longer crRNA spacers were also reported for a Type I-F system from Shewanella putrefaciens , however, shortening of spacer rendered effector complexes inactive (19).…”

CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production

“…Although spacer sequences in this CRISPR system are ~30 bp, it has previously been shown that ~10 bp are typically trimmed from the 5′ end of the spacer during crRNA-tracrRNA processing by RNAse III, yielding functional spacers of ~20 bp [47]. It is possible, however, that certain crRNAs retain the full length spacer as shown possible in the Type I-E CRISPR system from E. coli [58], but the effect that this would have on dCas9-mediated transcriptional repression of distinct targets in E. coli is currently unknown.…”