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CRISPR adaptation biases explain preference for acquisition of foreign DNA

Abstract

CRISPR–Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA (‘spacers’) are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

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Figure 1: Chromosome-scale hotspots for spacer acquisition.
Figure 2: Dependence of spacer acquisition on replication.
Figure 3: Chi sites define boundaries of protospacer hotspots.
Figure 4: Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns.
Figure 5: A model explaining the preference for foreign DNA in spacer acquisition.

Accession codes

Primary accessions

Sequence Read Archive

Data deposits

RNA sequencing data are available in the National Center for Biotechnology Information Sequence Read Archive database under accession numbers SRX862155SRX862158 in study SRP053013. Raw data of spacer sequences are accessible at http://www.weizmann.ac.il/molgen/Sorek/files/CRISPR_adaptation_2015/crispr_adaptation_2015_data.html.

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Acknowledgements

We thank N. Barkai, A. Tanay, E. Mick, S. Doron, A. Stern, T. Dagan and M. Shamir for discussion. We also thank the Skarstad group for providing the MG1655dnaC2 strain, the Michel group for providing the JJC1819 strain and D. Dar for assistance in Illumina sequencing. R.S. was supported, in part, by the Israel Science Foundation (personal grant 1303/12 and I-CORE grant 1796), the European Research Council Starting Grant programme (grant 260432), Human Frontier Science Program (grant RGP0011/2013), the Abisch-Frenkel foundation, the Pasteur-Weizmann Council, the Minerva Foundation, and by a Deutsch-Israelische Projektkooperation grant from the Deutsche Forschungsgemeinschaft. U.Q. was supported, in part, by the European Research Council Starting Grant programme (grant 336079), the Israel Science Foundation (grant 268/14) and the Israeli Ministry of Health (grant 9988-3). A.L. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship.

Author information

Affiliations

Authors

Contributions

M.G., U.Q., A.L. and R.S. conceived and designed the research studies; M.G., I.Y., O.A., M.M., G.A. and R.E. performed the experiments; A.L., M.G., U.Q. and R.S. analysed data; A.L., M.G., U.Q. and R.S. wrote the manuscript.

Corresponding authors

Correspondence to Udi Qimron or Rotem Sorek.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacers.

DNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1–Cas2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to wild-type (WT) arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location and source of newly acquired spacers.

Extended Data Figure 2 PAMs and DNA content along the E. coli BL21-AI genome.

a, Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10 kb. b, DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (GenBank accession number NC_012947). Areas where few or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).

Extended Data Figure 3 Distribution of newly acquired spacers on the genome during synchronized replication.

E. coli K-12ΔcasCdnaC2 cells were transferred from 39 °C (replication restrictive temperature) to 30 °C (replication permissive). Cas1–Cas2 were induced in these cells 30 min before the transfer to 30 °C and during the growth in 30 °C. Newly acquired spacers were sequenced at the given time points: a, following 20 min; b, following 40 min; c, following 60 min from replication initiation. The positions of the newly acquired spacers in windows of 100 kb are shown, and their fraction out of the total new spacers in the sample.

Extended Data Figure 4 A model explaining the preference for spacer acquisition near TerC compared with TerA in E. coli BL21-AI.

The DNA manipulation at the CRISPR region forms a replication fork stalling site, and leads to extensive spacer acquisition upstream of the CRISPR. While the clockwise fork is stalled at the CRISPR, the anticlockwise fork reaches the Ter region and is stalled at the respective Ter site, TerC, leading to extensive spacer acquisition upstream of TerC. Another factor that can contribute to the observed TerC/TerA bias may be that the clockwise replichore in E. coli (oriC to TerA) is longer than the anticlockwise one (oriC to TerC), leading the forks to stall at TerC more often than at TerA.

Extended Data Figure 5 The protein product of T7 gene 5.9 inhibits spacer acquisition activity.

E. coli BL21-AI strains harbouring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% l-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.

Extended Data Figure 6 Distribution of protospacers across the plasmids.

a, Distribution across pCtrl-Chi; b, distribution across pChi plasmids. Circular representation of the 4.7 kb plasmid is presented, with the inserted 4-Chi cluster present at the top of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar, 100,000 spacers. Pooled protospacers from two replicates are presented for each panel.

Extended Data Table 1 Spacer acquisition in normal and perturbed conditions
Extended Data Table 2 Replication-dependent spacer acquisition
Extended Data Table 3 Involvement of the DNA repair machinery in spacer acquisition

Supplementary information

Supplementary Table 1

This file contains the data for Supplementary Table 1. (XLSX 657 kb)

Supplementary Table 2

This table contains the bacterial strains, plasmids and oligonucleotides used in this study. (XLSX 16 kb)

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Levy, A., Goren, M., Yosef, I. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015). https://doi.org/10.1038/nature14302

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