AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing.

CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however, targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an "AddTag" sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene-editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing

Assessing biosynthetic potential of agricultural groundwater through metagenomic sequencing: A diverse anammox community dominates nitrate-rich groundwater.

BackgroundClimate change produces extremes in both temperature and precipitation causing increased drought severity and increased reliance on groundwater resources. Agricultural practices, which rely on groundwater, are sensitive to but also sources of contaminants, including nitrate. How agricultural contamination drives groundwater geochemistry through microbial metabolism is poorly understood.MethodsOn an active cow dairy in the Central Valley of California, we sampled groundwater from three wells at depths of 4.3 m (two wells) and 100 m (one well) below ground surface (bgs) as well as an effluent surface water lagoon that fertilizes surrounding corn fields. We analyzed the samples for concentrations of solutes, heavy metals, and USDA pathogenic bacteria of the Escherichia coli and Enterococcus groups as part of a long term groundwater monitoring study. Whole metagenome shotgun sequencing and assembly revealed taxonomic composition and metabolic potential of the community.ResultsElevated nitrate and dissolved organic carbon occurred at 4.3m but not at 100m bgs. Metagenomics confirmed chemical observations and revealed several Planctomycete genomes, including a new Brocadiaceae lineage and a likely Planctomycetes OM190, as well novel diversity and high abundance of nano-prokaryotes from the Candidate Phyla Radiation (CPR), the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea (DPANN) and the Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK) superphyla. Pathway analysis suggests community interactions based on complimentary primary metabolic pathways and abundant secondary metabolite operons encoding antimicrobials and quorum sensing systems.ConclusionsThe metagenomes show strong resemblance to activated sludge communities from a nitrogen removal reactor at a wastewater treatment plant, suggesting that natural bioremediation occurs through microbial metabolism. Elevated nitrate and rich secondary metabolite biosynthetic capacity suggest incomplete remediation and the potential for novel pharmacologically active compounds

Maternal IgG and IgA Antibodies Dampen Mucosal T Helper Cell Responses in Early Life

To maintain a symbiotic relationship between the host and its resident intestinal microbiota, appropriate mucosal T cell responses to commensal antigens must be established. Mice acquire both IgG and IgA maternally; the former has primarily been implicated in passive immunity to pathogens while the latter mediates host-commensal mutualism. Here, we report the surprising observation that mice generate T cell-independent and largely Toll-like receptor (TLR)-dependent IgG2b and IgG3 antibody responses against their gut microbiota. We demonstrate that maternal acquisition of these antibodies dampens mucosal T follicular helper responses and subsequent germinal center B cell responses following birth. This work reveals a feedback loop whereby T cell-independent, TLR-dependent antibodies limit mucosal adaptive immune responses to newly acquired commensal antigens and uncovers a broader function for maternal IgG

The pdm3 locus is a hotspot for recurrent evolution of female-limited color dimorphism in Drosophila

Sex-limited polymorphisms are an intriguing form of sexual dimorphism that offer unique opportunities to reconstruct the evolutionary changes that decouple male and female traits encoded by a shared genome. We investigated the genetic basis of a Mendelian female-limited color dimorphism (FLCD) that segregates in natural populations of more than 20 species of the Drosophila montium subgroup. In these species, females have alternative abdominal color morphs, light and dark, whereas males have only one color morph in each species. A comprehensive molecular phylogeny of the montium subgroup supports multiple origins of FLCD. Despite this, we mapped FLCD to the same locus in four distantly related species—the transcription factor POU domain motif\ua03 (pdm3), which acts as a repressor of abdominal pigmentation in D.\ua0melanogaster. In D.\ua0serrata, FLCD maps to a structural variant in the first intron of pdm3; however, this variant is not found in the three other species—D.\ua0kikkawai, D.\ua0leontia, and D.\ua0burlai—and sequence analysis strongly suggests the pdm3 alleles responsible for FLCD originated independently at least three times. We propose that cis-regulatory changes in pdm3 form sexually dimorphic and monomorphic alleles that segregate within species and are preserved, at least in one species, by structural variation. Surprisingly, pdm3 has not been implicated in the evolution of sex-specific pigmentation outside the montium subgroup, suggesting that the genetic paths to sexual dimorphism may be constrained within a clade but variable across clades

Assessing biosynthetic potential of agricultural groundwater through metagenomic sequencing: A diverse anammox community dominates nitrate-rich groundwater

<div><p>Background</p><p>Climate change produces extremes in both temperature and precipitation causing increased drought severity and increased reliance on groundwater resources. Agricultural practices, which rely on groundwater, are sensitive to but also sources of contaminants, including nitrate. How agricultural contamination drives groundwater geochemistry through microbial metabolism is poorly understood.</p><p>Methods</p><p>On an active cow dairy in the Central Valley of California, we sampled groundwater from three wells at depths of 4.3 m (two wells) and 100 m (one well) below ground surface (bgs) as well as an effluent surface water lagoon that fertilizes surrounding corn fields. We analyzed the samples for concentrations of solutes, heavy metals, and USDA pathogenic bacteria of the <i>Escherichia coli</i> and <i>Enterococcus</i> groups as part of a long term groundwater monitoring study. Whole metagenome shotgun sequencing and assembly revealed taxonomic composition and metabolic potential of the community.</p><p>Results</p><p>Elevated nitrate and dissolved organic carbon occurred at 4.3m but not at 100m bgs. Metagenomics confirmed chemical observations and revealed several Planctomycete genomes, including a new Brocadiaceae lineage and a likely Planctomycetes OM190, as well novel diversity and high abundance of nano-prokaryotes from the Candidate Phyla Radiation (CPR), the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea (DPANN) and the Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK) superphyla. Pathway analysis suggests community interactions based on complimentary primary metabolic pathways and abundant secondary metabolite operons encoding antimicrobials and quorum sensing systems.</p><p>Conclusions</p><p>The metagenomes show strong resemblance to activated sludge communities from a nitrogen removal reactor at a wastewater treatment plant, suggesting that natural bioremediation occurs through microbial metabolism. Elevated nitrate and rich secondary metabolite biosynthetic capacity suggest incomplete remediation and the potential for novel pharmacologically active compounds.</p></div