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Research Article | Ecological and Evolutionary Science

Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches

Yanbin Yin, Bowen Yang, Sarah Entwistle
Marcus J. Claesson, Editor
Yanbin Yin
Nebraska Food for Health Center, Department of Food Science and Technology, University of Nebraska—Lincoln, Lincoln, Nebraska, USA
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Bowen Yang
Nebraska Food for Health Center, Department of Food Science and Technology, University of Nebraska—Lincoln, Lincoln, Nebraska, USA
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Sarah Entwistle
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, USA
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Marcus J. Claesson
University College Cork
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DOI: 10.1128/mSystems.00455-19
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Figures

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  • FIG 1
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    FIG 1

    Genomic context of 45 experimentally characterized Acr representative genes. In total, 38 genomic loci are shown. Each locus contains at least one Acr gene (red arrows). There are 26 loci that also contain Aca genes (cyan arrows) with Pfam HTH or DUF1870 domains. Seven AcrIIA genes do not have neighboring Aca genes. The five AcrVA genes form four loci that also contain AcrIF genes, an AcrIC gene, and functional unknown genes (gray arrows) but no Aca genes. The plots were made with the Gene Graphics server (38). *, AcrF11 homologs were found to be next to other HTH domain-containing proteins as well, which were named Aca4 through Aca7 in reference 11. The archaeal viral AcrID1 has an upstream HTH protein, which has never been officially named and is named Aca8 here.

  • FIG 2
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    FIG 2

    Bioinformatics pipeline to identify genomic loci containing Acr homologs and/or Aca homologs. Guilt-by-association (GBA) was used to identify new HTH proteins (Aca proteins) that neighbor homologs of known Acr proteins in the RefSeq bacterial genomes and IMG/VR metagenome-assembled viral contigs (MACs). These Aca proteins were combined with published Aca proteins (Text S1) to search for more Aca homologs. GBA was further used to identify genomic loci (or operons) that contain Aca homologs. More filters were then used to select loci that are from genomes with annotated mobile genetic elements (MGEs), complete CRISPR-Cas systems, and self-targeting CRISPR spacers. For Diamond search (39), the –more-sensitive option was used to improve search sensitivity. For hmmsearch (http://hmmer.org/), the default parameters were used.

  • FIG 3
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    FIG 3

    Homologs of known Acr families in RefSeq and IMG/VR databases. (A) Breakdown numbers of homologs in each known Acr family (total of 975 for RefSeq and 2,022 for IMG/VR). (B) Breakdown numbers of homologs in each known Acr family that have neighboring Aca homologs (total of 224 for RefSeq and 165 for IMG/VR [Fig. 2]). Note that AcrIIA1 itself is considered an Aca; therefore, the two RefSeq AcrIIA1 genes in panel B coexist with other AcrIIA1 homologs in the same operons.

  • FIG 4
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    FIG 4

    One hundred seventy-three Acr homologs are from genomes with self-targeting CRISPR-Cas spacers of different subtypes. The y axis shows 26 known Acr families, which have homologs in genomes with self-targeting CRISPR-Cas spacers. The Cas subtypes of the self-targeting spacers (according to the work of Watters et al. [12]) are shown on the x axis. The numbers in the circles are the numbers of Acr homologs.

  • FIG 5
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    FIG 5

    Eight hundred seventeen putative Acr-Aca loci are found in 14 bacterial classes and inferred to target 10 CRISPR-Cas subtypes. The bubble plot shows the phylum-class versus the CRISPR-Cas subtype of the 817 Acr-Aca loci supported by the existence of self-targeting spacers in the genomes. The circle size and color are coded in proportion to the number of loci in that circle (in logarithmic scale). The subtype of Acr-Aca loci is inferred from the CRISPR-Cas self-targeting spacers reported in the paper by Watters et al. (12).

Tables

  • Figures
  • Supplemental Material
  • TABLE 1

    Genomic loci with Aca homologs and with Acr plus Aca homologs

    ParameterValue by filter steps in Fig. 2
    1, 21, 2, 31, 2, 3, 41, 2, 4
    No. of loci with Aca homologs53,2161,544508817
        No. of genomes29,3651,310478672
        No. (%) of genomes with >1 locus14,405 (49.1)184 (14.0)20 (4.2)97 (14.4)
        No. (%) of genomes with self-targeting spacers672 (2.3)478 (36.5)478 (100)672 (100)
    No. (%) of loci with Aca + Acr homologs1,193 (2.2)527 (34.1)367 (72.2)376 (46.0)
        No. (%) of genomes1,102 (3.8)511 (39.0)363 (75.9)370 (55.1)
        No. (%) of genomes with >1 locus81 (7.4)13 (2.5)4 (1.1)6 (1.6)
        No. (%) of genomes with self-targeting spacers370 (33.6)363 (71.0)363 (100)370 (100)
        No. (%) of Acr homologs (unique IDs)224 (100)95 (42.4)55 (24.6)62 (27.7)
        No. (%) of Aca homologs (unique IDs)157 (100)66 (42.0)39 (24.8)45 (28.7)
  • TABLE 2

    Sixteen experimentally characterized Acr-Aca loci in bacterial genomes

    Bacterial speciesRefSeq genome IDPublished Acr-Aca lociPresence of complete CRISPR-Cas
    and self-targeting spacer
    Listeria monocytogenes J0161GCF_000168635AcrIIA2-AcrIIA1Yes
    Listeria monocytogenes serotype 7 strain SLCC2482GCF_000210795AcrIIA2-AcrIIA3-AcrIIA1Yes
    Listeria monocytogenes J0161GCF_000168635AcrIIA4-AcrIIA1Yes
    Pseudomonas otitidisGCF_900111835AcrIE5-Aca1Yes
    Pseudomonas aeruginosa WH-SGI-V-07059GCF_001450485aAcrIF4-AcrIE3-Aca1No self-target spacer
    Haemophilus parainfluenzaeGCF_001053575aAcrIIC4-Aca2No self-target spacer
    Pseudomonas citronellolisGCF_001654435aAcrIE4-IF7-Aca1No self-target spacer
    Pseudomonas aeruginosa strain C 1426GCF_000412555aAcrIF11-Aca1No self-target spacer
    Pseudomonas aeruginosa strain Jp54GCF_003836565bAcrIF6-Aca1No self-target spacer
    Vibrio parahaemolyticusGCF_000736335aAcrIF9-Aca1No complete CRISPR-Cas
    Shewanella xiamenensisGCF_000712635aAcrIF10-Aca1No complete CRISPR-Cas
    Neisseria meningitidisGCF_001066195aAcrIIC1-Aca3No complete CRISPR-Cas
    Neisseria meningitidis 992008GCF_000724735aAcrIIC2-AcaIIC3-Aca3No complete CRISPR-Cas
    Simonsiella muelleri ATCC 29453GCF_000163775aAcrIIC5-Aca2No complete CRISPR-Cas
    Pseudomonas aeruginosa strain S708_C14_RSGCF_002136415bAcrIE6-Aca1No complete CRISPR-Cas
    Pseudomonas aeruginosa strain 359GCF_002312455bAcrIF12-Aca4No complete CRISPR-Cas
    • ↵a These genomes are contained in the version of the RefSeq database that we have mined but have no self-targeting spacers according to the work of Watters et al. (12).

    • ↵b These genomes were not in the version of the RefSeq database that we have mined, so we downloaded them separately from NCBI. All these genomes were analyzed using CRISPRCasFinder to confirm the presence of complete CRISPR-Cas systems and using our in-house programs to confirm the presence of self-targeting spacers.

  • TABLE 3

    Fifty-nine Acr-Aca loci with conflicting subtype assignments based on homology to known Acrs and on self-targeting spacers

    No. of lociaHomology to known AcrsSelf-targeting
    CRISPR-Cas
    subtype
    No. of unique
    Acr homolog IDs
    42AcrIF3I-E1
    4AcrIF6I-E1
    2AcrIF1I-E1
    2AcrIE3I-F1
    6AcrIIA2I-B1
    1AcrIIA3, AcrIIA2I-B1
    1AcrIIA4I-B1
    1AcrIE3, AcrIF4I-C1
    • ↵a Details about these loci can be found in Data Set S1.

  • TABLE 4

    Four putative Acr-Aca loci from four genomes with self-targeting spacers

    ParameterData for species namea:
    Pseudoramibacter
    alactolyticus
    ATCC 23263
    Bacteroides
    salyersiae strain
    2789STDY5608871
    Geoalkalibacter
    subterraneus
    Clostridioides
    difficile isolate
    VL_0239
    RefSeq genome IDGCF_000185505GCF_001405695GCF_000827125GCF_900013625
    Self-targeting
    CRISPR-Cas subtype
    III-AIII-BIV-AV-U4
    Acr-Aca locus on the same
    contig as spacer target
    WP_006597765.1 to
    WP_006597766.1
    WP_025819230.1 to
    WP_005928638.1
    WP_040200104.1 to
    WP_040200103.1
    WP_003438035.1 to
    WP_003438037.1
    Aca/HTH protein IDWP_006597766.1WP_005928638.1WP_040200103.1WP_003438035.1
    Aca is antitoxin hitYesYesYesYes
    Acr protein IDWP_006597765.1WP_025819230.1WP_040200104.1WP_003438037.1
    Acr is toxin hitNoYesYesNo
    No. of other
    candidate Acr-Aca loci
    0004
    Other CRISPR-Cas system(s)I-C, II-ANoI-CI-B
    • ↵a Details about these loci can be found in Data Set S3.

Supplemental Material

  • Figures
  • Tables
  • TEXT S1

    Supplementary results. Download Text S1, DOCX file, 0.3 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    Acr homolog counts in RefSeq and IMG/VR databases. Download Table S1, XLSX file, 0.02 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    Taxonomic distribution of 975 Acr homologs in RefSeq. Download Table S2, XLSX file, 0.01 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S3

    Habitat distribution of 2,022 Acr homologs in IMG/VR. Download Table S3, XLSX file, 0.01 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S4

    Pfam domain analysis of proteins surrounding the 975 Acr homologs in RefSeq. Download Table S4, XLSX file, 0.01 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S5

    Pfam domain analysis of proteins surrounding the 2,022 Acr homologs in IMG/VR. Download Table S5, XLSX file, 0.01 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • DATA SET S1

    One thousand one hundred ninety-three RefSeq genomic loci with both Acr and Aca homologs. Download Data Set S1, XLS file, 1.7 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • DATA SET S2

    One hundred fifty-five IMG/VR genomic loci with both Acr and Aca homologs. Download Data Set S2, XLS file, 0.3 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S6

    Taxonomic distribution of 224 Acr homologs in RefSeq that have neighboring Aca homologs. Download Table S6, XLSX file, 0.01 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • DATA SET S3

    Eight hundred seventeen RefSeq genomic loci from genomes with self-targeting spacers. Download Data Set S3, XLS file, 1.1 MB.

    Copyright © 2019 Yin et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches
Yanbin Yin, Bowen Yang, Sarah Entwistle
mSystems Sep 2019, 4 (5) e00455-19; DOI: 10.1128/mSystems.00455-19

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Bioinformatics Identification of Anti-CRISPR Loci by Using Homology, Guilt-by-Association, and CRISPR Self-Targeting Spacer Approaches
Yanbin Yin, Bowen Yang, Sarah Entwistle
mSystems Sep 2019, 4 (5) e00455-19; DOI: 10.1128/mSystems.00455-19
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KEYWORDS

Aca
Acr
CRISPR self-targeting
CRISPR-Cas
anti-CRISPR
gene neighborhood
genomic island
helix-turn-helix
phage
prophage

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