A cryptic plasmid is among the most numerous genetic elements in the human gut.

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states.

Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

Bacteriophages benefit from mobilizing pathogenicity islands encoding immune systems against competitors.

Bacteria encode sophisticated anti-phage systems that are diverse and versatile and display high genetic mobility. How this variability and mobility occurs remains largely unknown. Here, we demonstrate that a widespread family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs), carry an impressive arsenal of defense mechanisms, which can be disseminated intra- and inter-generically by helper phages. These defense systems provide broad immunity, blocking not only phage reproduction, but also plasmid and non-cognate PICI transfer. Our results demonstrate that phages can mobilize PICI-encoded immunity systems to use them against other mobile genetic elements, which compete with the phages for the same bacterial hosts. Therefore, despite the cost, mobilization of PICIs may be beneficial for phages, PICIs, and bacteria in nature. Our results suggest that PICIs are important players controlling horizontal gene transfer and that PICIs and phages establish mutualistic interactions that drive bacterial ecology and evolution.

Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders.

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1 retrotransposition; 80% of the larger inversions are balanced and affect twice as many nucleotides as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or retrotransposons. Since flanking repeats promote non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7 × 10-4 per locus per generation. Recurrent inversions exhibit a sex-chromosomal bias and co-localize with genomic disorder critical regions. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes specific haplotypes to disease-causing CNVs.

Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

Regulation of Division and Differentiation of Plant Stem Cells.

A major challenge in developmental biology is unraveling the precise regulation of plant stem cell maintenance and the transition to a fully differentiated cell. In this review, we highlight major themes coordinating the acquisition of cell identity and subsequent differentiation in plants. Plant cells are immobile and establish position-dependent cell lineages that rely heavily on external cues. Central players are the hormones auxin and cytokinin, which balance cell division and differentiation during organogenesis. Transcription factors and miRNAs, many of which are mobile in plants, establish gene regulatory networks that communicate cell position and fate. Small peptide signaling also provides positional cues as new cell types emerge from stem cell division and progress through differentiation. These pathways recruit similar players for patterning different organs, emphasizing the modular nature of gene regulatory networks. Finally, we speculate on the outstanding questions in the field and discuss how they may be addressed by emerging technologies.

Modularity and diversity of target selectors in Tn7 transposons.

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end.

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

Mobile Genetic Element Flexibility as an Underlying Principle to Bacterial Evolution.

Mobile genetic elements are key to the evolution of bacteria and traits that affect host and ecosystem health. Here, we use a framework of a hierarchical and modular system that scales from genes to populations to synthesize recent findings on mobile genetic elements (MGEs) of bacteria. Doing so highlights the role that emergent properties of flexibility, robustness, and genetic capacitance of MGEs have on the evolution of bacteria. Some of their traits can be stored, shared, and diversified across different MGEs, taxa of bacteria, and time. Collectively, these properties contribute to maintaining functionality against perturbations while allowing changes to accumulate in order to diversify and give rise to new traits. These properties of MGEs have long challenged our abilities to study them. Implementation of new technologies and strategies allows for MGEs to be analyzed in new and powerful ways.

Exploring the role of phage plasmids in gene transfers.

Bacteriophages and plasmids drive horizontal gene transfer (HGT) in bacteria. Phage-plasmids (P-Ps) are hybrids of plasmid and phages. Pfeifer and Rocha recently demonstrated that P-Ps can serve as intermediates in gene exchanges between these two types of elements, identified categories of preferentially transferred genes, and reconstructed gene flows involving phage P1-like P-Ps.

The Landscape of L1 Retrotransposons in the Human Genome Is Shaped by Pre-insertion Sequence Biases and Post-insertion Selection.

L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements.

Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition.

Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.

The evolutionary genomics of adaptation to stress in wild rhizobium bacteria.

Microbiota comprise the bulk of life's diversity, yet we know little about how populations of microbes accumulate adaptive diversity across natural landscapes. Adaptation to stressful soil conditions in plants provides seminal examples of adaptation in response to natural selection via allelic substitution. For microbes symbiotic with plants however, horizontal gene transfer allows for adaptation via gene gain and loss, which could generate fundamentally different evolutionary dynamics. We use comparative genomics and genetics to elucidate the evolutionary mechanisms of adaptation to physiologically stressful serpentine soils in rhizobial bacteria in western North American grasslands. In vitro experiments demonstrate that the presence of a locus of major effect, the nre operon, is necessary and sufficient to confer adaptation to nickel, a heavy metal enriched to toxic levels in serpentine soil, and a major axis of environmental soil chemistry variation. We find discordance between inferred evolutionary histories of the core genome and nreAXY genes, which often reside in putative genomic islands. This suggests that the evolutionary history of this adaptive variant is marked by frequent losses, and/or gains via horizontal acquisition across divergent rhizobium clades. However, different nre alleles confer distinct levels of nickel resistance, suggesting allelic substitution could also play a role in rhizobium adaptation to serpentine soil. These results illustrate that the interplay between evolution via gene gain and loss and evolution via allelic substitution may underlie adaptation in wild soil microbiota. Both processes are important to consider for understanding adaptive diversity in microbes and improving stress-adapted microbial inocula for human use.

Rapid dissemination of host metabolism-manipulating genes via integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

WHO elements - A new category of selfish genetic elements at the borderline between homing elements and transposable elements.

Homing genetic elements are a form of selfish DNA that inserts into a specific target site in the genome and spreads through the population by a process of biased inheritance. Two well-known types of homing element, called inteins and homing introns, were discovered decades ago. In this review we describe WHO elements, a newly discovered type of homing element that constitutes a distinct third category but is rare, having been found only in a few yeast species so far. WHO elements are inferred to spread using the same molecular homing mechanism as inteins and introns: they encode a site-specific endonuclease that cleaves the genome at the target site, making a DNA break that is subsequently repaired by copying the element. For most WHO elements, the target site is in the glycolytic gene FBA1. WHO elements differ from inteins and homing introns in two fundamental ways: they do not interrupt their host gene (FBA1), and they occur in clusters. The clusters were formed by successive integrations of different WHO elements into the FBA1 locus, the result of an 'arms race' between the endonuclease and its target site. We also describe one family of WHO elements (WHO10) that is no longer specifically associated with the FBA1 locus and instead appears to have become transposable, inserting at random genomic sites in Torulaspora globosa with up to 26 copies per strain. The WHO family of elements is therefore at the borderline between homing genetic elements and transposable elements.

The sound of silence: mechanisms and implications of HUSH complex function.

The vertebrate genome is under constant threat of invasion by genetic parasites. Whether the host can immediately recognize and respond to invading elements has been unclear. The discovery of the human silencing hub (HUSH) complex, and the finding that it provides immediate protection from genome invasion by silencing products of reverse transcription, have important implications for mammalian genome evolution. In this review, we summarize recent insights into HUSH function and describe how cellular introns provide a novel means of self-nonself discrimination, allowing HUSH to recognize and transcriptionally repress a broad range of intronless genetic elements. We discuss how HUSH contributes to genome evolution, and highlight studies reporting the critical role of HUSH in development and implicating HUSH in the control of immune signaling and cancer progression.

CRISPR enables heritable genome editing in planta.

Recent exciting developments in clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing showcase its potential to rapidly and efficiently edit genomes in planta, eliminating long processes of tissue culture and extensive breeding for crop improvement. These new methods offer heritable transgene-free edits in one generation, making them an attractive option for improving commercially important crops.

Heterogeneous changes in mobility in response to the SARS-CoV-2 Omicron BA.2 outbreak in Shanghai.

The coronavirus disease 2019 (COVID-19) pandemic and the measures taken by authorities to control its spread have altered human behavior and mobility patterns in an unprecedented way. However, it remains unclear whether the population response to a COVID-19 outbreak varies within a city or among demographic groups. Here, we utilized passively recorded cellular signaling data at a spatial resolution of 1 km × 1 km for over 5 million users and epidemiological surveillance data collected during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 outbreak from February to June 2022 in Shanghai, China, to investigate the heterogeneous response of different segments of the population at the within-city level and examine its relationship with the actual risk of infection. Changes in behavior were spatially heterogenous within the city and population groups and associated with both the infection incidence and adopted interventions. We also found that males and individuals aged 30 to 59 y old traveled more frequently, traveled longer distances, and their communities were more connected; the same groups were also associated with the highest SARS-CoV-2 incidence. Our results highlight the heterogeneous behavioral change of the Shanghai population to the SARS-CoV-2 Omicron BA.2 outbreak and the effect of heterogenous behavior on the spread of COVID-19, both spatially and demographically. These findings could be instrumental for the design of targeted interventions for the control and mitigation of future outbreaks of COVID-19, and, more broadly, of respiratory pathogens.