Critical roles for ‘housekeeping’ nucleases in type III CRISPR-Cas immunity
- Microbiology Department, University of Illinois Urbana-Champaign, United States
Figures
Figure 1 with 1 supplement
RNase R and PNPase are necessary for crRNA maturation in the cell.
(A) The type III-A CRISPR-Cas system (herein referred to as CRISPR-Cas10) in S. epidermidis RP62a encodes three spacers (colored squares), four repeats (light gray squares), and nine …
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Figure 1—source data 1
Raw uncropped image for panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig1-data1-v2.zip
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Figure 1—source data 2
Raw uncropped image for panel E.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig1-data2-v2.zip
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Figure 1—source data 3
Percent intermediate crRNAs for individual replicates in panel F.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Confirmation of rnr knock-out and knock-in S. epidermidis strains.
(A) Illustration of the rnr genomic locus and corresponding primers used to amplify the gene (see Supplementary file 2 for primer sequences). (B) PCR products of rnr region from representative S. …
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Figure 1—figure supplement 1—source data 1
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Raw uncropped image for panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig1-figsupp1-data2-v2.zip
Figure 2 with 1 supplement
RNase R and PNPase are sufficient to complete crRNA maturation in a purified system.
(A) Illustration of experimental flow of the crRNA maturation nuclease assay. P, PNPase; R, RNase R; C, Cbf1; Cas10-Csm (71), Cas10-Csm complexes purified from S. epidermidis LM1680ΔpnpΔrnr. (B) …
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Figure 2—source data 1
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig2-data1-v2.zip
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Figure 2—source data 2
Raw uncropped image for panel C.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig2-data2-v2.zip
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Figure 2—source data 3
Raw uncropped image for panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig2-data3-v2.zip
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Figure 2—source data 4
Percent intermediate crRNAs for individual replicates in panel E.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
RNase R alone cannot complete crRNA maturation in a purified system.
Total crRNAs associated with purified Cas10-Csm (71) complexes are shown after digestion with indicated nucleases for different timepoints (15, 30, and 60 min). CrRNAs were extracted using TRIzol …
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Figure 2—figure supplement 1—source data 1
Raw uncropped image.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig2-figsupp1-data1-v2.zip
Figure 3 with 2 supplements
Csm5 interacts with RNase R.
(A) Purified recombinant WT Csm5 is shown. The protein was resolved in an SDS-PAGE gel and visualized using Coomassie G-250 staining. M, denaturing protein marker; kDa, kilodalton. See Figure …
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Figure 3—source data 1
Raw uncropped image for panel A.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-data1-v2.zip
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Figure 3—source data 2
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-data2-v2.zip
Figure 3—figure supplement 1
Csm5 does not interact with bovine serum albumin (BSA).
Shown is a native gel in which BSA was resolved alone or with increasing concentrations of Csm5 WT. The gel shown is a representative of three independent trials. NM, native protein marker; kDa, …
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Figure 3—figure supplement 1—source data 1
Raw uncropped image.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Csm5 interacts weakly with RNase R in a pulldown assay.
(A) Illustration of a pulldown assay in which Csm5-His10-Smt3 (input 1) is loaded onto a Ni2+-agarose column, the column is washed to remove unbound protein, and then untagged RNase R or protein …
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Figure 3—figure supplement 2—source data 1
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-figsupp2-data1-v2.zip
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Figure 3—figure supplement 2—source data 2
Raw uncropped image for panel C.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-figsupp2-data2-v2.zip
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Figure 3—figure supplement 2—source data 3
Raw uncropped image for panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig3-figsupp2-data3-v2.zip
Figure 4 with 1 supplement
A predicted disordered region in Csm5 promotes crRNA maturation.
(A) Illustration showing the distribution of charged residues, predicted disordered regions, and truncations introduced in Csm5. Positions of charged residues (positive, cyan; negative, magenta) are …
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Figure 4—source data 1
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig4-data1-v2.zip
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Figure 4—source data 2
Raw uncropped image for panel C.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig4-data2-v2.zip
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Figure 4—source data 3
Percent intermediate crRNAs for individual replicates in panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig4-data3-v2.xlsx
Figure 4—figure supplement 1
Predicted disordered regions of Csm5.
(A) Predicted disordered regions in S. epidermidis Csm5 are shown. The PONDR (Predictor of Natural Disordered Regions) score was derived from the web-based tool with the same name (pondr.com). …
Figure 5 with 1 supplement
Csm5 interacts with and stimulates PNPase via a predicted disordered region.
(A) Purified recombinant Csm5Δ46 is shown, in which IDR2 has been deleted. The protein was resolved on an SDS-PAGE gel and visualized using Coomassie G-250 staining. M, denaturing protein marker; …
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Figure 5—source data 1
Raw uncropped image for panel A.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig5-data1-v2.zip
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Figure 5—source data 2
Raw uncropped image for panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig5-data2-v2.zip
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Figure 5—source data 3
Raw uncropped image for panel C.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig5-data3-v2.zip
Figure 5—figure supplement 1
Csm5Δ46 retains interaction with RNase R.
Shown is a native gel in which RNase R was resolved alone or with increasing concentrations of Csm5Δ46. The gel shown is a representative of three independent trials. NM, native protein marker; kDa, …
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Figure 5—figure supplement 1—source data 1
Raw uncropped image.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
RNase R and PNPase work synergistically to promote robust anti-plasmid immunity.
(A) Illustration of the anti-plasmid assay is shown in which the conjugative plasmid pG0400 is transferred from a S. aureus RN4220 donor (not shown) into various S. epidermidis RP62a recipient …
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Figure 6—source data 1
Recipients, transconjugants, and conjugation efficiencies for independent replicates in panel C.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig6-data1-v2.xlsx
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Figure 6—source data 2
Recipients, transconjugants, and conjugation efficiencies for independent replicates in panel E.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
RNase R and PNPase are dispensable for anti-phage immunity.
(A) Illustration of the assay used to test anti-phage immunity in S. epidermidis LM1680. In this assay, dilutions of the siphophage CNPx are placed atop lawns of cells containing variants of pcrispr-…
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Figure 6—figure supplement 1—source data 1
Phage plaque counts for individual replicates in panel B.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig6-figsupp1-data1-v2.xlsx
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Figure 6—figure supplement 1—source data 2
Phage plaque counts for individual replicates in panel D.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig6-figsupp1-data2-v2.xlsx
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Figure 6—figure supplement 1—source data 3
Phage plaque counts for individual replicates in panel F.
- https://cdn.elifesciences.org/articles/81897/elife-81897-fig6-figsupp1-data3-v2.xlsx
Figure 7
A model for how diverse housekeeping nucleases are enlisted to ensure successful defense.
(A) During Cas10-Csm complex assembly, Csm5 recruits and/or stimulates RNase R and PNPase through direct interactions. The unprotected 3′-ends of intermediate crRNAs are trimmed as a consequence of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Staphylococcus epidermidis) | cbf1 | NA | GenBank: CP000029.1_SERP1378 | Encodes Cbf1 |
| Gene (S. epidermidis) | rnr | NA | GenBank: CP000029.1_SERP0450 | Encodes RNase R |
| Gene (S. epidermidis) | pnp | NA | GenBank: CP000029.1_SERP0841 | Encodes PNPase |
| Gene (S. epidermidis) | csm5 | NA | GenBank: CP000029.1_SERP2457 | Encodes Csm5 |
| Strain, strain background (Staphylococcus aureus, RN4220) | RN4220 | PMID:21378186 | GenBank: NZ_AFGU00000000 | LA Marraffini (Rockefeller University) |
| Strain, strain background (S. epidermidis, RP62a) | RP62a | PMID:3679536 | GenBank: CP000029.1 | LA Marraffini (Rockefeller University) |
| Strain, strain background (S. epidermidis, LAM104) | Δspc1-3 | PMID:19095942 | LA Marraffini (Rockefeller University), derivative of RP62a with crispr deletion | |
| Strain, strain background (S. epidermidis, LM1680) | LM1680 | PMID:24086164 | LA Marraffini (Rockefeller University), derivative of RP62a with large deletion | |
| Strain, strain background (phage Andhra) | Andhra | PMID:28357414 | GenBank: KY442063 | Isolated in-house |
| Strain, strain background (phage ISP) | ISP | PMID:21931710 | GenBank: FR852584 | LA Marraffini (Rockefeller University) |
| Strain, strain background (phage CNPx) | CNPx | PMID:26755632 | GenBank: NC_031241 | LA Marraffini (Rockefeller University) |
| Genetic reagent (Staphylococcus epidermidis, RP62a) | RP62a Δpnp | PMID:30942690 | Created in-house | |
| Genetic reagent (S. epidermidis, RP62a) | RP62a Δrnr | This paper | The central 2316 nucleotides of the rnr coding region are deleted, see Figure 1—figure supplement 1 | |
| Genetic reagent (S. epidermidis, RP62a) | RP62a Δrnr::rnr* | This paper | A copy of rnr with two silent mutations reintroduced into the rnr locus, see Figure 1—figure supplement 1 | |
| Genetic reagent (S. epidermidis, RP62a) | RP62a Δrnr Δpnp | This paper | Contains in-frame deletions of rnr (described in the cells above) and pnp (PMID:30942690) | |
| Genetic reagent (S. epidermidis, RP62a) | RP62a Δrnr Δpnp::rnr* | This paper | A copy of rnr with two silent mutations reintroduced into the rnr locus, see Figure 1—figure supplement 1 | |
| Genetic reagent (S. epidermidis, LM1680) | LM1680 Δpnp | PMID:30942690 | Created in-house | |
| Genetic reagent (S. epidermidis, LM1680) | LM1680 Δrnr | This paper | The central 2316 nucleotides of the rnr coding region are deleted, see Figure 1—figure supplement 1 | |
| Genetic reagent (S. epidermidis, LM1680) | LM1680 Δrnr::rnr* | This paper | A copy of rnr with two silent mutations reintroduced into the rnr locus, see Figure 1—figure supplement 1 | |
| Genetic reagent (S. epidermidis, LM1680) | LM1680 Δrnr Δpnp | This paper | Contains in-frame deletions of rnr (described in the cells above) and pnp (PMID:30942690) | |
| Genetic reagent (S. epidermidis, LM1680) | LM1680 Δrnr Δpnp::rnr* | This paper | A copy of rnr with two silent mutations reintroduced into the rnr locus, see Figure 1—figure supplement 1 | |
| Recombinant DNA reagent | pKOR1 | PMID:16051359 | LA Marraffini (Rockefeller University) | |
| Recombinant DNA reagent | pKOR1-Δrnr | This paper | To create in-frame deletion of rnr via allelic replacement | |
| Recombinant DNA reagent | pKOR1-rnr* | This paper | To create complementation of rnr with silent mutations via allelic replacement | |
| Recombinant DNA reagent | pcrispr-cas | PMID:23935102 | LA Marraffini (Rockefeller University) | |
| Recombinant DNA reagent | pcrisprcas/csm5H6NΔ18 | This paper | Contains 18 amino acids deletion encompassing IDR2 in Csm5 | |
| Recombinant DNA reagent | pcrisprcas/csm5H6NΔ31 | This paper | Contains 31 amino acids deletion encompassing IDR2 in Csm5 | |
| Recombinant DNA reagent | pcrisprcas/csm5H6NΔ46 | This paper | Contains 46 amino acids deletion encompassing IDR2 in Csm5 | |
| Recombinant DNA reagent | pET28b-H10Smt3-csm5 | PMID:28204542 | Created in-house | |
| Recombinant DNA reagent | pET28b-H10Smt3-csm5Δ46 | This paper | Contains 46 amino acids deletion encompassing IDR2 in Csm5; for overexpression and purification of Csm5Δ46 | |
| Sequence-based reagent | 5'-ACGAGAACAC GUAUGCCGA AGUAUAUAAAUC | Eurofins MWG Operon | A 31-nt single-stranded RNA substrate for nuclease assays, see Figure 5 | |
| Sequence-based reagent | DNA oligonucleotides (multiple) | Eurofins MWG Operon | To build and sequence recombinant DNA constructs, see Supplementary file 2 | |
| Sequence-based reagent | Decade Markers System | Fisher Scientific | Cat# AM7778 | |
| Peptide, recombinant protein | EcoRI | New England Biolabs | Cat# R0101S | |
| Peptide, recombinant protein | T4 Polynucleotide kinase | New England Biolabs | Cat# M0201L | |
| Peptide, recombinant protein | T4 DNA Ligase | New England Biolabs | Cat# M0202S | |
| Peptide, recombinant protein | DpnI | New England Biolabs | Cat# R0176S | |
| Peptide, recombinant protein | Lysostaphin | AmbiProducts via Fisher | Cat# NC0318863 | |
| Peptide, recombinant protein | Pierce Protease and Phosphatase Inhibitor Mini Tablets | Thermo Fisher | Cat# 88669 | |
| Peptide, recombinant protein | SUMO Protease | MCLAB, http://www.mclab.com/SUMO-Protease.html | Cat# SP-100 | |
| Peptide, recombinant protein | Bovine serum albumin (BSA) | VWR | Cat# 97061-420 | |
| Peptide, recombinant protein | PageRuler Plus Prestained Protein Ladder, 10–250 kDa | Thermo Fisher | Cat# 26619 | |
| Peptide, recombinant protein | NativeMark Unstained Protein Standard | Invitrogen via Thermo Fisher | Cat# LC0725 | |
| Commercial assay or kit | EZNA Cycle Pure Kit | Omega Bio-tek via VWR | Cat# 101318-892 | |
| Commercial assay or kit | EZNA Plasmid DNA Mini Kit | Omega Bio-tek via VWR | Cat# 101318-898 | |
| Commercial assay or kit | Advance Centrifugal Devices 10K MWCO | Pall via VWR | Cat# 89131-980 | |
| Commercial assay or kit | Disposable Gravity Flow Columns for Protein Purification | G-Biosciences via VWR | Cat# 82021-346 | |
| Commercial assay or kit | G-25 Spin Columns | IBI Scientific via VWR | Cat# IB06010 | |
| Chemical compound or drug | HisPur Ni-NTA Resin | Thermo Fisher | Cat# 88222 | |
| Chemical compound or drug | TRIzol Reagent | Thermo Fisher | Cat#15596026 | |
| Chemical compound or drug | g-32P-ATP | PerkinElmer | Cat# BLU502H250UC | |
| Software, algorithm | ImageQuant TL | GE Healthcare/Life Sciences | RRID:SCR_014246 | Version 8.2, used for densitometry |
| Software, algorithm | PONDR | Molecular Kinetics, Inc, Washington State University and the WSU Research Foundation | pondr.com | Used to predict disordered regions in Csm5 |
| Software, algorithm | PyMOL | The PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC | RRID:SCR_000305 | Version 2.5, used for structural analyses |
| Software, algorithm | CRISPR-Cas10 Protospacer Selector Tool | ahatoum/CRISPR-Cas10- Protospacer-Selector is licensed under the GNU General Public License v3.0 (Bari et al., 2017) | https://github.com/ahatoum/CRISPR-Cas10-Protospacer-Selector | Used to predict protospacer sequence to target phage Andhra. |
Additional files
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Supplementary file 1
Accompanies Figures 2, 3 and 5, Figure 3—figure supplement 1, and Figure 5—figure supplement 1.
Theoretical molecular weights and isoelectric points of purified proteins in this study.
- https://cdn.elifesciences.org/articles/81897/elife-81897-supp1-v2.docx
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Supplementary file 2
DNA oligonucleotides used for cloning and PCR in this study.
- https://cdn.elifesciences.org/articles/81897/elife-81897-supp2-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/81897/elife-81897-mdarchecklist1-v2.docx
