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Abstract Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming “CandidatusMethanoperedens” archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of hostMethanoperedens, indicating that Borgs augment theMethanoperedensactivity in situ. We reconstructed the first complete 4.00 Mbp genome for aMethanoperedensthat is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enableMethanoperedensto distinguish their genomes from those of Borgs. Very high Borg toMethanoperedensratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells. 

Abstract CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity¹. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient^2–5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation^6,7, but many CRISPR–Cas systems lack Cas4⁸. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited^9,10exonucleases for faithful acquisition of new CRISPR immune sequences. 

Abstract CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1—Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference. 

ABSTRACT Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.