Molecular determinants for CRISPR RNA maturation in the Cas10–Csm complex and roles for non-Cas nucleases

Abstract

CRISPR–Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR–Cas system that encodes the Cas10–Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10–Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10–Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10–Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR–Cas immunity and central nucleic acid metabolism.

Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features

“…Similarly, Staphylococcus epidermidis (type III-A) has crRNAs that are 37, 43, and 72 nt in length (50). Walker et al (28) reported that 39 trimming of crRNAs in the type III-A S. epidermidis (Csm) complex is catalyzed by a trans-acting non-Cas nuclease, probably polyribonucleotide phosphorylase. However, mature crRNAs detected here in the M. tuberculosis system were of uniform size (;71 nt; Figs.…”

“…Accumulating evidence has demonstrated that crRNA biogenesis depends critically on the activity of the Cas6 superfamily of endonucleases in type I and III CRISPR/Cas systems (6). In type III-A systems, Cas6 typically cleaves pre-crRNA to generate intermediate crRNAs that are then incorporated into the crRNP complex where the 39 end sequences may be trimmed by polyribonucleotide phosphorylase (28). Here, to verify that Cas6 is involved in crRNA processing in M. tuberculosis as in other species, we constructed a Cas6 deletion mutant (Dcas6); as expected, crRNA generation was not detected (Supplemental Fig.…”

Expression and Purification of the Cas10-Csm Complex from Staphylococci

“…The overexpression and purification of recombinant CRISPR-associated proteins from Escherichia coli (both the Cas10-Csm complex and individual subunits) followed by in vitro biochemical assays have revealed important insights into their functions (Hatoum-Aslan et al ., 2013; Samai et al ., 2015; Walker et al ., 2016). However, such assays fail to 1) recover information about protein function and stability in the native cellular environment, and 2) identify biologically relevant binding partners that are not a part of the core Cas10-Csm complex.…”

“…However, such assays fail to 1) recover information about protein function and stability in the native cellular environment, and 2) identify biologically relevant binding partners that are not a part of the core Cas10-Csm complex. Indeed, purification of Cas10-Csm from its native S. epidermidis background has yielded additional insights into the genetic requirements for complex stability and function, crRNA processing, and non-Cas binding partners that might play a role in the CRISPR-Cas pathway (Hatoum-Aslan et al ., 2013 and 2014; Walker et al ., 2016). Here, we provide a detailed protocol for the purification of Cas10-Csm from S. epidermidis or S. aureus strains bearing the Type III-A CRISPR-Cas system on a plasmid.…”

“…The method consists of a two-step purification protocol involving Ni 2+ -affinity chromatography and a DNA affinity biotin pull-down, which together yield a pure preparation of the Cas10-Csm complex. This approach has been used previously to analyze the effects of mutations on Cas10-Csm complex integrity (Hatoum-Aslan et al ., 2014), crRNA formation (Hatoum-Aslan et al ., 2013), and to detect binding partners that directly interact with the core Cas10-Csm complex (Walker et al ., 2016). Importantly, this approach can be easily adapted for use in other Staphylococcus species to probe and understand their native Type III-A CRISPR-Cas systems.…”