Abstract
CRISPR–Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5′ end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5′ end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5′ end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.
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Data availability
Sequencing data generated for this study have been deposited in the NCBI Sequence Read Archive (SRA) under the BioProjectID PRJNA901420. Source data are provided with this paper.
Code availability
Custom python scripts were generated to quantify read coverage patterns and dinucleotide frequencies, and to parse and reformat alignment output files for display on the UCSC Genome Browser. The codes are available from the corresponding authors upon request or S.C.G. at garrett@uchc.edu.
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Acknowledgements
We thank the members of the Terns, Graveley and Santangelo labs for the helpful discussions. M.P.T. discloses support for this work from the National Institutes of Health (R35GM118160). B.R.G. discloses support for this work from the National Institutes of Health (R35GM118140). T.J. Santangelo discloses support for this work from the National Institutes of Health (R35GM143963 and RO1GM100329).
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E.A.W., S.C.G. and R.J.C. conducted experiments, analysed and interpreted results and wrote the manuscript. L.M.C., T.J. Sanders., C.J.M., B.R.W., R.L.V., R.F. and B.R. conducted experiments and interpreted results. T.J. Santangelo., B.R.G. and M.P.T. supervised experiments, interpreted results and wrote the manuscript and/or guided its conception and completion.
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Extended data
Extended Data Fig. 1 Steps of integration.
Cartoon diagram shows the steps by which a spacer is integrated into the CRISPR array at the leader-adjacent repeat.
Extended Data Fig. 2 Distributions of aligned DNA sequences from MNase protection assay.
MNase protected DNA (Fig. 1a) was sequenced by HTS, aligned, and genome browser tracks were generated to examine the genome-wide distribution of aligned DNA fragments. Overlapping subtracks are color-coded according to the size of the DNA fragment (red: 30 bp +/- 5; golden yellow: 60 bp +/- 5; green: 90 bp +/- 5; blue 120 bp +/- 5). Positions of CRISPR loci are indicated (leader in pale yellow, repeat array in black). Approximately 10 kb of genome space upstream and downstream from each CRISPR locus is shown. Six replicates (three stationary growth, three exponential) were sequenced and yielded similar patterns; a representative replicate is shown here.
Extended Data Fig. 3 Positions of MNase protected DNA fragments and promoter elements within leaders of the seven CRISPR loci in P. furiosus.
Genome browser tracks were examined to determine the distribution of aligned MNase-protected DNA fragments within CRISPR leaders. Overlapping subtracks are color-coded according to the size of the DNA fragment (red: 30 bp +/- 5; golden yellow: 60 bp +/- 5; green: 90 bp +/- 5; blue 120 bp +/- 5). The x-axis indicates the cumulative depth of read coverage. Six replicates (three stationary growth (St), three exponential (Ex)) are shown. We noted that the 60 bp read coverage (peak height) over L1 and LR was unusually high compared to 60 bp coverage elsewhere in the genome. To quantify this, we analyzed sliding 60 bp windows across the genome, determined the density of 60 bp reads in those sliding windows, then binned and tallied the results to determine the distribution of 60 bp read densities. The values found under the label “60mer peak height” indicate the percentile of the L1 or LR peaks for each replicate; for example, the LR peak in CR1 exponential replicate 1 is higher than 99.92% of all 60 bp windows in the genome. We also noted that L1 and LR peaks usually had more 60mer coverage than 90mer coverage, even though 90mer reads were more abundant elsewhere in the genome. We used the sliding windows to quantify the 60mer to 90mer read density ratios across the genome. The values found under the label “60/90 peak ratio” indicate how biased towards 60mer read covereager the L1 or LR peak is; for example, the LR peak in CR1 exponential replicate 1 is more biased towards 60mer read coverage than 97.68% of all 60 bp windows in the genome.
Extended Data Fig. 4 Nucleotide alignment of seven CRISPR array leaders with MNase protected DNA peaks, promoter elements, and integration sites mapped.
Nucleotide sequences of seven leaders were aligned; conserved nucleotide regions are highlighted in gray. Peaks of MNase protected DNA are highlighted in yellow; the purple star and line at the leader-repeat junction show the position of typical, in vivo spacer integration. Promoter elements in the leader are labeled (BRE and TATA).
Extended Data Fig. 5 Growth, RNA expression, and histone abundance characteristics in wildtype, ∆histone A, and ∆histone B P. furiosus strains used in this study.
Total, ribo-depleted RNA was sequenced for wildtype, ∆histone A, and ∆histone B strains (five replicates each) and analyzed using the DESeq2 package to identify annotated transcripts with differential expression between (a) wildtype and ∆histone A and (b) wildtype and ∆histone B. Volcano plots show raw p-values against log2-fold changes in abundance for each annotated transcript. A difference in transcript abundance was considered significant if the log2-fold difference was equal or greater than 0.58 and the adjusted p-value was equal or less than 0.1; significantly different points are shown in blue; all other points for transcripts not meeting this threshold are colored gray. (c) Wildtype, ∆histone A, and ∆histone B strains were grown in liquid medium supplemented with pyruvate and Na2S at 95 °C and optical density (OD600nm) was measured every hour as a proxy for cell growth. Blank sample contained medium but no inoculum. Error bars show standard error of the mean for three biological replicates. (d) Western blots, employing polyclonal antibodies raised against HTkA (that recognize HTkA, HTkB, HPfA, and HPfB), were done on total protein from each strain harvested either from exponential (E, 10 hour time point) or stationary (S, 30 hour time point) phase cells. Recombinantly purified HPfA and HPfB were resolved on the gels to provide size references. Six biological replicates were run with similar results; a representative blot image is shown here. Although samples were boiled in 2% SDS before gel loading, histones retained dimer, tetramer, and larger multimeric complexes, giving rise to the multi-band ladder appearance visible on the gel.
Extended Data Fig. 6 Conservation of histones in Euryarchaeota and purification of recombinant proteins.
(a) Clustal Omega Multiple Sequence Alignment of histones from P. furiosus (Pfu A and Pfu B), Thermococcus kodakarensis (Tko A and Tko B), and Methanothermus fervidus (HMf A and HMf B). * = completely conserved residues,: = conservation between groups with strongly similar properties,. = conservation between groups with weakly similar properties. (b) SDS PAGE gels with the purified proteins used in the in vitro integration assays. Cas1 = 37.5 kDa, Cas2 = 10 kDa, P. furiosus histones A = 7.4 kDa and B = 7.3 kDa, T. kodakarensis histones A = 7.3 kDa and B = 7.1 kDa, TrmBL2 = 30.6 kDa.
Extended Data Fig. 7 In vitro evaluation of spacer integration into four CRISPR arrays in the presence or absence of P. furiosus histones.
(a) Gel images show representative results from PCR (carried out as in Fig. 5d) with primers targeting four CRISPR arrays: CRISPR5, CRISPR6, CRISPR7, and CRISPR8. The expected sizes for PCR products resulting from integrations at repeats 1 - 5 are marked with R1 – R5, respectively. Red asterisks identify the band corresponding to integrations at repeat 1, which is the natural, preferred point of integration in vivo. (b) Intensity of all PCR bands was quantified using ImageJ and the proportion of integration events at the five repeats was determined. Single biological replicates were evaluated with two different primer pairs producing with similar outcomes; one PCR reaction is shown and quantitated here.
Extended Data Fig. 8 MNase protection assay to characterize binding patterns for P. furiosus histones incubated with the pCR7-long plasmid.
(a) Gel image of DNA fragments generated by micrococcal nuclease digestion of pCR7-long plasmid incubated with purified recombinant P. furiosus histones A and B. Colored boxes highlight major bands of protected DNA. The gel image is for a digest containing 100 units of MNase; a 500 unit digestion was also done and resulting DNA bands were much fainter. (b) DNA bands highlighted in A were sequenced and genome browser tracks were generated. Overlapping subtracks are color-coded according to the size of the DNA fragment (red: 30 bp +/- 5; golden yellow: 60 bp +/- 5; green: 90 bp +/- 5; blue 120 bp +/- 5). The full CRISPR7 and part of one repeat are shown, with the purple line indicating the position where integration would typically occur in vivo. The x-axis indicates the cumulative depth of read coverage. One replicate was done for each treatment (100 and 500 units MNase).
Extended Data Fig. 9 In vitro evaluation of spacer integration into pCR7-long in the presence or absence of DNA-binding proteins.
(a) Diagrammatric representation of the PCR used to assess in vitro integration into pCR7-long. (b) Gel images show representative results from the PCR in (a) when recombinant purified histones, Alba, or Trmbl2 were added to the reaction at the indicated concentrations. The expected sizes for PCR products resulting from integrations at repeats 1 - 4 are marked with R1 - R4, respectively. Red asterisks identify the band corresponding to integrations at repeat 1, which is the natural, preferred point of integration in vivo. (c) Intensity of all PCR bands was quantified using ImageJ and the proportion of integration events at the five repeats was determined. Three replicates were assessed with similar outcomes, representative gels and quantifications are shown here.
Extended Data Fig. 10 Unbiased HTS sequencing of in vitro integration of a spacer into pCR7-long.
The in vitro integration assay with and without histones (a) or alba (b) was carried out, as before, and integration events were then sequenced using a two-step, semi-degenerate PCR protocol which targets all DNA fragments bearing the spacer. Sequencing results were used to generate genome browser tracks; the amplitude of the black peaks in these tracks indicate the total number of reads supporting an integration event at that nucleotide position. The positions of the leader (pale yellow) and repeats (black) are shown. Assay products with histones were sequenced for six biological replicates yielding very similar patterns; with alba one biological replicate was sequenced.
Supplementary information
Source data
Source Data Fig. 1
Uncropped agarose gel images and MNase digested DNA.
Source Data Fig. 3
Uncropped agarose gel images and PCR products.
Source Data Fig. 4
Uncropped agarose gel images and PCR products.
Source Data Fig. 5
Uncropped agarose gel images and PCR products.
Source Data Extended Data Fig. 5
Uncropped gel image and western blot.
Source Data Extended Data Fig. 6
Uncropped gel images and western blot.
Source Data Extended Data Fig. 7
Uncropped agarose gel images and PCR products.
Source Data Extended Data Fig. 8
Uncropped agarose gel images and MNase digested DNA.
Source Data Extended Data Fig. 9
Uncropped agarose gel images and PCR products.
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Watts, E.A., Garrett, S.C., Catchpole, R.J. et al. Histones direct site-specific CRISPR spacer acquisition in model archaeon. Nat Microbiol 8, 1682–1694 (2023). https://doi.org/10.1038/s41564-023-01446-3
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DOI: https://doi.org/10.1038/s41564-023-01446-3
