CRISPR-associated proteins 1 and 2 (Cas1–2) are necessary and sufficient for new spacer acquisition in some CRISPR-Cas systems (e.g., type I-E), but adaptation in other systems (e.g., type II-A) involves the crRNA-guided surveillance complex. Here we show that the type I-F Cas1–2/3 proteins are necessary and sufficient to produce low levels of spacer acquisition, but the presence of the type I-F crRNA-guided surveillance complex (Csy) improves the efficiency of adaptation and significantly increases the fidelity of protospacer adjacent motif selection. Sequences selected for integration are preferentially derived from specific regions of extrachromosomal DNA, and patterns of spacer selection are highly reproducible between independent biological replicates. This work helps define the role of the Csy complex in I-F adaptation and reveals that actively replicating mobile genetic elements have antigenic signatures that facilitate their integration during CRISPR adaptation.
Get full access to this article
View all access options for this article.
References
1.
BarrangouR, FremauxC, DeveauH, et al.CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315:1709–1712. DOI: 10.1126/science.1138140.
2.
CadyKC, Bondy-DenomyJ, HeusslerGE, et al.The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J Bacteriol. 2012; 194:5728–5738. DOI: 10.1128/JB.01184-12.
3.
ErdmannS, Le Moine BauerS, GarrettRA. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol Microbiol. 2014; 91:900–917. DOI: 10.1111/mmi.12503.
4.
JacksonSA, McKenzieRE, FagerlundRD, et al.CRISPR-Cas: adapting to change. Science. 2017; 356:eaal5056. DOI: 10.1126/science.aal5056.
5.
KlompeSE, SternbergSH. Harnessing “A Billion Years of Experimentation”: the ongoing exploration and exploitation of CRISPR-Cas immune systems. CRISPR J. 2018; 1:141–158. DOI: 10.1089/crispr.2018.0012.
6.
LevyA, GorenMG, YosefI, et al.CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature. 2015; 520:505–510. DOI: 10.1038/nature14302.
7.
YosefI, GorenMG, QimronU. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012; 40:5569–5576. DOI: 10.1093/nar/gks216.
8.
LiM, WangR, ZhaoD, et al.Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Res. 2014; 42:2483–2492. DOI: 10.1093/nar/gkt1154.
9.
LiuT, LiuZ, YeQ, et al.Coupling transcriptional activation of CRISPR-Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus. Nucleic Acids Res. 2017; 45:8978–8992. DOI: 10.1093/nar/gkx612.
10.
HelerR, SamaiP, ModellJW, et al.Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature. 2015; 519:199–202. DOI: 10.1038/nature14245.
11.
WeiY, TernsRM, TernsMP. Cas9 function and host genome sampling in Type II-A CRISPR-Cas adaptation. Genes Dev. 2015; 29:356–361. DOI: 10.1101/gad.257550.114.
12.
MojicaFJ, Diez-VillasenorC, Garcia-MartinezJ, et al.Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009; 155:733–740. DOI: 10.1099/mic.0.023960-0.
13.
VorontsovaD, DatsenkoKA, MedvedevaS, et al.Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery. Nucleic Acids Res. 2015; 43:10848–10860. DOI: 10.1093/nar/gkv1261.
14.
RichterC, GristwoodT, ClulowJS, et al.In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype IF CRISPR/Cas system. PLoS One. 2012; 7:e49549. DOI: 10.1371/journal.pone.0049549.
15.
RollinsMF, ChowdhuryS, CarterJ, et al.Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity. Proc Natl Acad Sci U S A. 2017; 114:E5113–E5121. DOI: 10.1073/pnas.1616395114.
16.
FagerlundRD, WilkinsonME, KlykovO, et al.Spacer capture and integration by a type I-F Cas1-Cas2–Cas3 CRISPR adaptation complex. Proc Natl Acad Sci U S A. 2017; 114:E5122–E5128. DOI: 10.1073/pnas.1618421114.
17.
AlmendrosC, GuzmanNM, Garcia-MartinezJ, et al.Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR-Cas I-F systems. Nat Microbiol. 2016; 1:16081. DOI: 10.1038/nmicrobiol.2016.81.
18.
DatsenkoKA, WannerBL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000; 97:6640–6645. DOI: 10.1073/pnas.120163297.
19.
RichterC, DyRL, McKenzieRE, et al.Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 2014; 42:8516–8526. DOI: 10.1093/nar/gku527.
20.
StaalsRH, JacksonSA, BiswasA, et al.Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR-Cas system. Nat Commun. 2016; 7:12853. DOI: 10.1038/ncomms12853.
21.
RollinsMF, ChowdhuryS, CarterJ, et al.Structure reveals a mechanism of CRISPR-RNA-guided nuclease recruitment and anti-CRISPR viral mimicry. Mol Cell. 2019; 74:132–142.e135. DOI: 10.1016/j.molcel.2019.02.001.
22.
McKenzieRE, AlmendrosC, VinkJNA, et al.Using CAPTURE to detect spacer acquisition in native CRISPR arrays. Nat Protoc. 2019; 14:976–990. DOI: 10.1038/s41596-018-0123-5.
23.
MorganM, AndersS, LawrenceM, et al.ShortRead: a bioconductor package for input, quality assessment and exploration of high-throughput sequence data. Bioinformatics. 2009; 25:2607–2608. DOI: 10.1093/bioinformatics/btp450.
24.
PagèsH AP, GentlemanR, DebRoyS. Biostrings: efficient manipulation of biological strings. R package version 2520. Bioconductor. 2019. DOI: 10.18129/B9.bioc.Biostrings (last accessed October13, 2020).
25.
KassambaraA. ggpubr: 'ggplot2' based publication ready plots. R package version 023. CRAN. 2019. https://CRAN.R-project.org/package=ggpubr (last accessed October13, 2020).
26.
WickhamH. ggplot2: elegant graphics for data analysis. R package version 321. Springer-Verlag NewYork. 2016. ISBN 978-3-319-24277-4. https://CRAN.R-project.org/package=ggplot2 (last accessed October13, 2020).
DillinghamMS, KowalczykowskiSC. RecBCD enzyme and the repair of double-stranded DNA breaks. Microbiol Mol Biol Rev. 2008; 72:642–671. DOI: 10.1128/MMBR.00020-08.
ChengKC, SmithGR. Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli. J Mol Biol. 1987; 194:747–750. DOI: 10.1016/0022-2836(87)90252-x.
33.
RozkovA, Avignone-RossaCA, ErtlPF, et al.Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence. Biotechnol Bioeng. 2004; 88:909–915. DOI: 10.1002/bit.20327.
34.
OwDSW, LeeDY, TungHH, et al. Plasmid regulation and systems-level effects on Escherichia coli metabolism. Syst Biol Biotechnol Escherichia Coli. 2009:273–294. DOI: 10.1007/978-1-4020-9394-4_14.
35.
KimS, LoeffL, ColomboS, et al.Selective loading and processing of prespacers for precise CRISPR adaptation. Nature. 2020; 579:141–145. DOI: 10.1038/s41586-020-2018-1.
36.
BudhathokiJB, XiaoY, SchulerG, et al.Real-time observation of CRISPR spacer acquisition by Cas1-Cas2 integrase. Nat Struct Mol Biol. 2020; 27:489–499. DOI: 10.1038/s41594-020-0415-7.
37.
GarrettS, ShiimoriM, WattsEA, et al.Primed CRISPR DNA uptake in Pyrococcus furiosus. Nucleic Acids Res. 2020; 48:6120–6135. DOI: 10.1093/nar/gkaa381.
38.
TangJ, AkerboomJ, VaziriA, et al.Near-isotropic 3D optical nanoscopy with photon-limited chromophores. Proc Natl Acad Sci U S A. 2010; 107:10068–10073. DOI: 10.1073/pnas.1004899107.
39.
YaoS, HelinskiDR, ToukdarianA. Localization of the naturally occurring plasmid ColE1 at the cell pole. J Bacteriol. 2007; 189:1946–1953. DOI: 10.1128/JB.01451-06.
40.
EdgarR, RokneyA, FeeneyMet al.Bacteriophage infection is targeted to cellular poles. Mol Microbiol. 2008; 68:1107–1116. DOI: 10.1111/j.1365-2958.2008.06205.x.
41.
JoyeuxM. Preferential localization of the bacterial nucleoid. Microorganisms. 2019; 7:204. DOI: 10.3390/microorganisms7070204.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
