Discovery of Next-Generation Antimicrobials through Bacterial Self-Screening of Surface-Displayed Peptide Libraries.

Peptides have great potential to combat antibiotic resistance. While many platforms can screen peptides for their ability to bind to target cells, there are virtually no platforms that directly assess the functionality of peptides. This limitation is exacerbated when identifying antimicrobial peptides because the phenotype, death, selects against itself and has caused a scientific bottleneck that confines research to a few naturally occurring classes of antimicrobial peptides. We have used this seeming dissonance to develop Surface Localized Antimicrobial Display (SLAY), a platform that allows screening of unlimited numbers of peptides of any length, composition, and structure in a single tube for antimicrobial activity. Using SLAY, we screened ∼800,000 random peptide sequences for antimicrobial function and identified thousands of active sequences, dramatically increasing the number of known antimicrobial sequences. SLAY hits present with different potential mechanisms of peptide action and access to areas of antimicrobial physicochemical space beyond what nature has evolved. VIDEO ABSTRACT.

GENTANGLE: integrated computational design of gene entanglements.

SUMMARY: The design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased stability. The design of functional overlapping gene pairs is a challenging procedure, and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high-performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user-specified gene pairs. AVAILABILITY AND IMPLEMENTATION: The GENTANGLE source code and its submodules are freely available on GitHub at https://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results and is freely available on GitHub at https://github.com/BiosecSFA/datangle. The GENTANGLE container is freely available on Singularity Cloud Library at https://cloud.sylabs.io/library/khyox/gentangle/gentangle.sif. The GENTANGLE repository wiki (https://github.com/BiosecSFA/gentangle/wiki), website (https://biosecsfa.github.io/gentangle/), and user manual contain detailed instructions on how to use the different components of software and data, including examples and reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html).

Prolonging genetic circuit stability through adaptive evolution of overlapping genes.

The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

Species divergence in gut-restricted bacteria of social bees.

Host-associated microbiomes, particularly gut microbiomes, often harbor related but distinct microbial lineages, but how this diversity arises and is maintained is not well understood. A prerequisite for lineage diversification is reproductive isolation imposed by barriers to gene flow. In host-associated microbes, genetic recombination can be disrupted by confinement to different hosts, for example following host speciation, or by niche partitioning within the same host. Taking advantage of the simple gut microbiome of social bees, we explore the diversification of two groups of gut-associated bacteria, Gilliamella and Snodgrassella, which have evolved for 80 million y with honey bees and bumble bees. Our analyses of sequenced genomes show that these lineages have diversified into discrete populations with limited gene flow. Divergence has occurred between symbionts of different host species and, in some cases, between symbiont lineages within a single host individual. Populations have acquired genes to adapt to specific hosts and ecological niches; for example, Gilliamella lineages differ markedly in abilities to degrade dietary polysaccharides and to use the resulting sugar components. Using engineered fluorescent bacteria in vivo, we show that Gilliamella lineages localize to different hindgut regions, corresponding to differences in their abilities to use spatially concentrated nitrogenous wastes of hosts. Our findings show that bee gut bacteria can diversify due to isolation in different host species and also due to spatial niche partitioning within individual hosts, leading to barriers to gene flow.

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

Obligate bacterial endosymbionts limit thermal tolerance of insect host species.

The thermal tolerance of an organism limits its ecological and geographic ranges and is potentially affected by dependence on temperature-sensitive symbiotic partners. Aphid species vary widely in heat sensitivity, but almost all aphids are dependent on the nutrient-provisioning intracellular bacterium Buchnera, which has evolved with aphids for 100 million years and which has a reduced genome potentially limiting heat tolerance. We addressed whether heat sensitivity of Buchnera underlies variation in thermal tolerance among 5 aphid species. We measured how heat exposure of juvenile aphids affects later survival, maturation time, and fecundity. At one extreme, heat exposure of Aphis gossypii enhanced fecundity and had no effect on the Buchnera titer. In contrast, heat suppressed Buchnera populations in Aphis fabae, which suffered elevated mortality, delayed development and reduced fecundity. Likewise, in Acyrthosiphon kondoi and Acyrthosiphon pisum, heat caused rapid declines in Buchnera numbers, as well as reduced survivorship, development rate, and fecundity. Fecundity following heat exposure is severely decreased by a Buchnera mutation that suppresses the transcriptional response of a gene encoding a small heat shock protein. Similarly, absence of this Buchnera heat shock gene may explain the heat sensitivity of Ap. fabae Fluorescent in situ hybridization revealed heat-induced deformation and shrinkage of bacteriocytes in heat-sensitive species but not in heat-tolerant species. Sensitive and tolerant species also differed in numbers and transcriptional responses of heat shock genes. These results show that shifts in Buchnera heat sensitivity contribute to host variation in heat tolerance.

Symbionts shape host innate immunity in honeybees.

The gut microbiome plays a critical role in the health of many animals. Honeybees are no exception, as they host a core microbiome that affects their nutrition and immune function. However, the relationship between the honeybee immune system and its gut symbionts is poorly understood. Here, we explore how the beneficial symbiont Snodgrassella alvi affects honeybee immune gene expression. We show that both live and heat-killed S. alvi protect honeybees from the opportunistic pathogen Serratia marcescens and lead to the expression of host antimicrobial peptides. Honeybee immune genes respond differently to live S. alvi compared to heat-killed S. alvi, the latter causing a more extensive immune expression response. We show a preference for Toll pathway upregulation over the Imd pathway in the presence of both live and heat-killed S. alvi. Finally, we find that live S. alvi aids in clearance of S. marcescens from the honeybee gut, supporting a potential role for the symbiont in colonization resistance. Our results show that colonization by the beneficial symbiont S. alvi triggers a replicable honeybee immune response. These responses may benefit the host and the symbiont, by helping to regulate gut microbial members and preventing overgrowth or invasion by opportunists.

Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont.

Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tn-seq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization. Comparison of Tn-seq results from laboratory cultures and from monoinoculated worker bees reveal that 519 of 2,226 protein-coding genes in S. alvi are essential in culture, whereas 399 are not essential but are beneficial for gut colonization. Genes facilitating colonization fall into three broad functional categories: extracellular interactions, metabolism, and stress responses. Extracellular components with strong fitness benefits in vivo include trimeric autotransporter adhesins, O antigens, and type IV pili (T4P). Experiments with T4P mutants establish that T4P in S. alvi likely function in attachment and biofilm formation, with knockouts experiencing a competitive disadvantage in vivo. Metabolic processes promoting colonization include essential amino acid biosynthesis and iron acquisition pathways, implying nutrient scarcity within the hindgut environment. Mechanisms to deal with various stressors, such as for the repair of double-stranded DNA breaks and protein quality control, are also critical in vivo. This genome-wide study identifies numerous genetic networks underlying colonization by a gut commensal in its native host environment, including some known from more targeted studies in other host-microbe symbioses.

One-step genome engineering in bee gut bacterial symbionts.

Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi. We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Escape mutants that alleviate this burden can rapidly evolve and take over cell populations, making genetic engineering less reliable and predictable. Synthetic biologists often use genetic parts encoded on plasmids, but their burden is rarely characterized. We measured how 301 BioBrick plasmids affected Escherichia coli growth and found that 59 (19.6%) were burdensome, primarily because they depleted the limited gene expression resources of host cells. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be unclonable. We made this model available online for education ( https://barricklab.org/burden-model ) and added our burden measurements to the iGEM Registry. Our results establish a fundamental limit on what DNA constructs and genetic modifications can be successfully engineered into cells.

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

Honey bee functional genomics using symbiont-mediated RNAi.

Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.

Single-step genome engineering in the bee gut symbiont Snodgrassella alvi.

Honey bees are economically relevant pollinators experiencing population declines due to a number of threats. As in humans, the health of bees is influenced by their microbiome. The bacterium Snodgrassella alvi is a key member of the bee gut microbiome and has a role in excluding pathogens. Despite this importance, there are not currently any easy-to-use methods for modifying the S. alvi chromosome to study its genetics. To solve this problem, we developed a one-step procedure that uses electroporation and homologous recombination, which we term SnODIFY (Snodgrassella-specific One-step gene Deletion or Insertion to alter FunctionalitY). We used SnODIFY to create seven single-gene knockout mutants and recovered mutants for all constructs tested. Nearly all transformants had the designed genome modifications, indicating that SnODIFY is highly accurate. Mutant phenotypes were validated through knockout of Type 4 pilus genes, which led to reduced biofilm formation. We also used SnODIFY to insert heterologous sequences into the genome by integrating fluorescent protein-coding genes. Finally, we confirmed that genome modification is dependent on S. alvi's endogenous RecA protein. Because it does not require expression of exogenous recombination machinery, SnODIFY is a straightforward, accurate, and lightweight method for genome editing in S. alvi. This workflow can be used to study the functions of S. alvi genes and to engineer this symbiont for applications including protection of honey bee health.

The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila -colonizing Orbaceae.

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli , a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying new host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species. IMPORTANCE: To fully understand how microbes have evolved to interact with their environments, one must be able to modify their genomes. However, it can be difficult and laborious to discover which genetic tools and approaches work for a new isolate. Bacteria from the recently described Orbaceae family are common in the microbiomes of insects. We developed the Pathfinder plasmid toolkit for testing the compatibility of different genetic parts with newly cultured bacteria. We demonstrate its utility by engineering Orbaceae strains isolated from flies to express fluorescent proteins and characterizing how they colonize the Drosophila melanogaster gut. Orbaceae are widespread in Drosophila in the wild but have not been included in laboratory studies examining how the gut microbiome affects fly nutrition, health, and longevity. Our work establishes a path for genetic studies aimed at understanding and altering interactions between these and other newly isolated bacteria and their hosts.

The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae.

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli, a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying microbial ecology and host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.

Prospects for probiotics in social bees.

Social corbiculate bees are major pollinators. They have characteristic bacterial microbiomes associated with their hives and their guts. In honeybees and bumblebees, worker guts contain a microbiome composed of distinctive bacterial taxa shown to benefit hosts. These benefits include stimulating immune and metabolic pathways, digesting or detoxifying food, and defending against pathogens and parasites. Stressors including toxins and poor nutrition disrupt the microbiome and increase susceptibility to opportunistic pathogens. Administering probiotic bacterial strains may improve the health of individual bees and of hives, and several commercial probiotics are available for bees. However, evidence for probiotic benefits is lacking or mixed. Most bacterial species used in commercial probiotics are not native to bee guts. We present new experimental results showing that cultured strains of native bee gut bacteria colonize robustly while bacteria in a commercial probiotic do not establish in bee guts. A defined community of native bee gut bacteria resembles unperturbed native gut communities in its activation of genes for immunity and metabolism in worker bees. Although many questions remain unanswered, the development of natural probiotics for honeybees, or for commercially managed bumblebees, is a promising direction for protecting the health of managed bee colonies. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Engineering insects from the endosymbiont out.

Insects are an incredibly diverse group of animals with species that benefit and harm natural ecosystems, agriculture, and human health. Many insects have consequential associations with microbes: bacterial symbionts may be embedded in different insect tissues and cell types, inherited across insect generations, and required for insect survival and reproduction. Genetically engineering insect symbionts is key to understanding and harnessing these associations. We summarize different types of insect-bacteria relationships and review methods used to genetically modify endosymbiont and gut symbiont species. Finally, we discuss recent studies that use this approach to study symbioses, manipulate insect-microbe interactions, and influence insect biology. Further progress in insect symbiont engineering promises to solve societal challenges, ranging from controlling pests to protecting pollinator health.

Field-Realistic Tylosin Exposure Impacts Honey Bee Microbiota and Pathogen Susceptibility, Which Is Ameliorated by Native Gut Probiotics.

Antibiotics have been applied to honey bee (Apis mellifera) hives for decades to treat Paenibacillus larvae, which causes American foulbrood disease and kills honey bee larvae. One of the few antibiotics approved in apiculture is tylosin tartrate. This study examined how a realistic hive treatment regimen of tylosin affected the gut microbiota of bees and susceptibility to a bacterial pathogen. Tylosin treatment reduced bacterial species richness and phylogenetic diversity and reduced the absolute abundances and strain diversity of the beneficial core gut bacteria Snodgrassella alvi and Bifidobacterium spp. Bees from hives treated with tylosin died more quickly after being fed a bacterial pathogen (Serratia marcescens) in the laboratory. We then tested whether a probiotic cocktail of core bee gut species could bolster pathogen resistance. Probiotic exposure increased survival of bees from both control and tylosin-treated hives. Finally, we measured tylosin tolerance of core bee gut bacteria by plating cultured isolates on media with different tylosin concentrations. We observed highly variable responses, including large differences among strains of both S. alvi and Gilliamella spp. Thus, probiotic treatments using cultured bee gut bacteria may ameliorate harmful perturbations of the gut microbiota caused by antibiotics or other factors. IMPORTANCE The antibiotic tylosin tartrate is used to treat honey bee hives to control Paenibacillus larvae, the bacterium that causes American foulbrood. We found that bees from tylosin-treated hives had gut microbiomes with depleted overall diversity as well as reduced absolute abundances and strain diversity of the beneficial bee gut bacteria Snodgrassella alvi and Bifidobacterium spp. Furthermore, bees from treated hives suffered higher mortality when challenged with an opportunistic pathogen. Bees receiving a probiotic treatment, consisting of a cocktail of cultured isolates of native bee gut bacteria, had increased survival following pathogen challenge. Thus, probiotic treatment with native gut bacteria may ameliorate negative effects of antibiotic exposure.

Engineered symbionts activate honey bee immunity and limit pathogens.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure.

Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of 5, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector, we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.