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

Abstract

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. Author summary The evolution and origins of viruses are long-standing questions in the field of biology. Viral genomes provide fundamental information to infer the evolution and origin of viruses. However, viruses are extraordinarily diverse, and there are no single genes shared across entire species. Several methods were developed to collect viral genomes from metagenome. To infer viral genomes from metagenome, previous approaches relied on reference viral genomes. We thought that such reference-based methods may not be sufficient to uncover diverse viral genomes; therefore, we developed a pipeline that utilizes CRISPR, a prokaryotic adaptive immunological memory. Using this pipeline, we discovered more than 10,000 positively complete CRISPR-targeted genomes from human gut metagenome datasets. A substantial portion of the discovered genomes encoded various types of capsid proteins, supporting the contention that these sequences are viral. Although the majority of these capsid-protein-coding sequences were previously characterized, we notably discovered Inoviridae genomes that were previously difficult to infer as being viral. Furthermore, some of the remaining unclassified sequences without a detectable capsid-protein-encoding gene had a notably low protein-coding ratio. Overall, our pipeline successfully discovered viruses and previously uncharacterized presumably mobile genetic elements targeted by CRISPR.