Comprehensive discovery of CRISPR-targeted terminally redundant sequences in the human gut metagenome: Viruses, plasmids, and more

PLoS Comput Biol. 2021 Oct 21;17(10):e1009428. doi: 10.1371/journal.pcbi.1009428. eCollection 2021 Oct.


Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse, and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes that characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores the memory of previous exposure. Our protocol can infer CRISPR-targeted sequences, including viruses, plasmids, and previously uncharacterized elements, and predict their hosts using unassembled short-read metagenomic sequencing data. By analyzing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences, which are likely complete circular genomes. The sequences included 2,154 tailed-phage genomes, together with 257 complete crAssphage genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 56 genomes of Inoviridae species, and 95 previously uncharacterized circular small genomes that have no reliably predicted protein-coding gene. We predicted the host(s) of approximately 70% of the discovered genomes at the taxonomic level of phylum by linking protospacers to taxonomically assigned CRISPR direct repeats. These results demonstrate that our protocol is efficient for de novo inference of CRISPR-targeted sequences and their host prediction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaea / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gastrointestinal Microbiome / genetics*
  • Humans
  • Metagenome / genetics*
  • Metagenomics
  • Plasmids / genetics*
  • Sequence Analysis, DNA
  • Viruses / genetics*

Grants and funding

R.S. is funded by the Research Organization of Information and Science ( L.N. is funded by JSPS Research Fellow DC1 21J22509 ( P.T.N is funded by MEXT Doctoral Scholarship 180243 ( J.I. is funded by JSPS KAKENHI JP20K15767 ( and JSPS Research Fellow PD 19J01713 ( I.I. is funded by JSPS KAKENHI 20K21405 ( The other authors received no specific funding for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.