A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

Thank you for submitting your article "A Type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity" for consideration by eLife. Your article has been reviewed by two peer reviewers and the evaluation has been overseen by a Reviewing Editor and Gisela Storz as the Senior Editor. The reviewers, who have opted to remain anonymous, have recommended your paper for publication, pending minor revisions.

Reviewer #1:

In this well-written manuscript, Lucy Chou-Zheng and Asma Hatoum-Aslan demonstrate that PNPase is required for efficient crRNA maturation in the type III-A system from S. epidermidis, and that both PNPase and RNase J2 play important roles in degrading foreign nucleic acids. While the function of Cas proteins in this system has received considerable attention, the importance of ancillary factors that complement CRISPR-Cas immune function have not been investigated with the same rigor. This paper adds considerable mechanistic insight and I anticipate that this work will inspire more careful examination of host factors in other immune systems. I have a few very minor suggestions.

Minor comments:

Introduction, first paragraph: Consider adding Yosef et al., 2012. I think this was the first experimental evidence of Cas protein mediated acquisition.

Subsection “RNaseJ2 is required for CRISPR-Cas10 mediated anti-plasmid and anti-phage immunity”, last paragraph, second sentence: I think this sentence is missing a word (i.e., might conditional).

Figure 3 legend: I did not see and explanation for the dashed line in Figure 3D and F.

Reviewer #2:

In this manuscript, the authors explore the synergistic activity of the type III-A CRISPR-Cas system and the degradasome complex, which is required for RNA processing and degradation in bacteria. In prior work, the authors provided evidence that several degradasome nucleases (among others, PNPase, RNase J1 and RNase J2) associate with the Csm-Cas10 complex, which mediates type III-A CRISPR-Cas immunity. Here, the authors demonstrate in Staphylococcus epidermidis that degradasome nucleases are important for CRISPR RNA (crRNA) maturation and clearance of phage nucleic acids during CRISPR-Cas immunity.

The authors establish three S. epidermidis strains (∆pnp, ∆rnjB and ∆pnp∆rnjB) in which either PNPase, RNase J2 or both are deleted (unfortunately the authors could not obtain a knockout of RNase J1, which may be an essential gene). They first tested the importance of these proteins for crRNA maturation and observed that crRNA intermediates accumulate in the absence of PNPase, while RNase J2 is dispensable for crRNA maturation. Next, the authors tested whether PNPase or RNase J2 are required for CRISPR-Cas-mediated immunity using both plasmid conjugation and phage challenge assays. They demonstrate that RNase J2 is required for efficient Csm-Cas10-mediated interference against plasmid conjugation. In a striking set of data, they also show that RNase J2 is required for Csm-Cas10-mediated immunity against phages, and that both PNPase and RNase J2 are required for efficient clearing of phage nucleic acids. Interestingly, they also show that both nucleases may be involved in clearing phage DNA (or in limiting the propagation of the DNA) even in the absence of the CRISPR-Cas system. To support the role of RNase J2, and its partner nuclease RNase J1, the authors performed in vitro RNA and DNA cleavage assays. They convincingly demonstrate that RNase J1 cleaves single-stranded DNA, and that this activity requires RNase J2. These data provide direct evidence to support a role for RNase J1/J2 in phage DNA clearance.

Overall, this is an exceptional study that demonstrates cooperation between a house-keeping complex and the specialized CRISPR-Cas machinery. Other studies have suggested that non-Cas nucleases, including RecBCD, RNase III and PNPase, may be involved in various steps of the immune system. This study provides further compelling evidence toward this emerging trend. The data are very well presented, the manuscript is easy to follow, and the results are reasonably interpreted. The experiments really present a tour-de-force, with the authors anticipating many of my questions. I have a few remaining questions that could likely be addressed through changes to the text.

Minor comments:

1) In Figure 2B, different crRNA intermediates appear to accumulate in the ∆pnp∆rnjB strain compared to the ∆pnp strain (running between the 71 and 43 nt band). The authors do not comment on these products. Was this cleavage pattern observed in all replicates? Do the authors have an idea of what these products might be? Although it may be premature to speculate on the origin of these intermediates, the authors should at least note this difference between the two strains in the text, especially if it was reproducible.

2) There is an apparent discrepancy in the data shown in Figure 3 that the authors do not comment on. Conjugation efficiency in the ∆pnp∆rnjB strain (2.2 x 10^-4) did not recover to WT levels in the absence of the pCRISPR-Cas plasmid (2.6 x 10^-3). This suggests that CRISPR-Cas activity is sufficient to provide a low level of plasmid interference (reduction of conjugation efficiency by an order of magnitude) even in the absence of PNPase and RNase J2. However, in the phage challenge assays, the ∆pnp∆rnjB strains displays similar phage sensitivity to strains lacking the CRISPR-Cas system, suggesting that the immune system is not sufficient to provide any immunity against phages in the absence of PNPase and RNase J2. The authors could comment on this apparent difference. Is this simply a consequence of differences in the two types of assays? Could this be related to the apparent CRISPR-independent activity of the degradasome nucleases against phage DNA shown in Figure 5—figure supplement 1?

3) Although the authors data convincingly demonstrate that RNase J2 (and likely J1) cooperate with the CRISPR-Cas machinery, there remains a possibility that deletion of degradasome proteins affects expression of other host factors that may be involved in CRISPR-mediated immunity. The authors should address this possibility in the text.