Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).

The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene ß-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of ß-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of ß-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out ß-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.

Genome-Wide Comparative Analysis of SRCR Gene Superfamily in Invertebrates Reveals Massive and Independent Gene Expansions in the Sponge and Sea Urchin.

Without general adaptative immunity, invertebrates evolved a vast number of heterogeneous non-self recognition strategies. One of those well-known adaptations is the expansion of the immune receptor gene superfamily coding for scavenger receptor cysteine-rich domain containing proteins (SRCR) in a few invertebrates. Here, we investigated the evolutionary history of the SRCR gene superfamily (SRCR-SF) across 29 metazoan species with an emphasis on invertebrates. We analyzed their domain architectures, genome locations and phylogenetic distribution. Our analysis shows extensive genome-wide duplications of the SRCR-SFs in Amphimedon queenslandica and Strongylocentrotus purpuratus. Further molecular evolution study reveals various patterns of conserved cysteines in the sponge and sea urchin SRCR-SFs, indicating independent and convergent evolution of SRCR-SF expansion during invertebrate evolution. In the case of the sponge SRCR-SFs, a novel motif with seven conserved cysteines was identified. Exon-intron structure analysis suggests the rapid evolution of SRCR-SFs during gene duplications in both the sponge and the sea urchin. Our findings across nine representative metazoans also underscore a heightened expression of SRCR-SFs in immune-related tissues, notably the digestive glands. This observation indicates the potential role of SRCR-SFs in reinforcing distinct immune functions in these invertebrates. Collectively, our results reveal that gene duplication, motif structure variation, and exon-intron divergence might lead to the convergent evolution of SRCR-SF expansions in the genomes of the sponge and sea urchin. Our study also suggests that the utilization of SRCR-SF receptor duplication may be a general and basal strategy to increase immune diversity and tissue specificity for the invertebrates.

Immune diversity in lophotrochozoans, with a focus on recognition and effector systems.

Lophotrochozoa is one of the most species-rich but immunologically poorly explored phyla. Although lack of acquired response in a narrow sense, lophotrochozoans possess various genetic mechanisms that enhance the diversity and specificity of innate immune system. Here, we review the recent advances of comparative immunology studies in lophotrochozoans with focus on immune recognition and effector systems. Haemocytes and coelomocytes are general important yet understudied player. Comparative genomics studies suggest expansion and functional divergence of lophotrochozoan immune reorganization systems is not as "homogeneous and simple" as we thought including the large-scale expansion and molecular divergence of pattern recognition receptors (PRRs) (TLRs, RLRs, lectins, etc.) and signaling adapters (MyD88s etc.), significant domain recombination of immune receptors (RLR, NLRs, lectins, etc.), extensive somatic recombination of fibrinogenrelated proteins (FREPs) in snails. Furthermore, there are repeatedly identified molecular mechanisms that generate immune effector diversity, including high polymorphism of antimicrobial peptides and proteins (AMPs), reactive oxygen and nitrogen species (RONS) and cytokines. Finally, we argue that the next generation omics tools and the recently emerged genome editing technicism will revolutionize our understanding of innate immune system in a comparative immunology perspective.

The Potential of Gut Microbiota Metabolic Capability to Detect Drug Response in Rheumatoid Arthritis Patients.

Gut microbiota plays an essential role in the development of rheumatoid arthritis (RA) and affects drug responses. However, the underlying mechanism remains elusive and urgent to elucidate to explore the pathology and clinical treatment of RA. Therefore, we selected methotrexate (MTX) as an example of RA drugs to explore the interactions between the gut microbiota and drug responses and obtain an in-depth understanding of their correlation from the perspective of the metabolic capability of gut microbiota on drug metabolism. We identified 2,654 proteins and the corresponding genes involved in MTX metabolism and then profiled their abundances in the gut microbiome datasets of four cohorts. We found that the gut microbiota harbored various genes involved in MTX metabolism in healthy individuals and RA patients. Interestingly, the number of genes involved in MTX metabolism was not significantly different between response (R) and non-response (NR) groups to MTX, but the gene composition in the microbial communities significantly differed between these two groups. Particularly, several models were built based on clinical information, as well as data on the gene, taxonomical, and functional biomarkers by using the random forest algorithm and then validated. Our findings provide bases for clinical management not only of RA but also other gut microbiome-related diseases. First, it suggests that the potential metabolic capability of gut microbiota on drug metabolism is important because they affect drug efficiency; as such, clinical treatment strategies should incorporate the gene compositions of gut microbial communities, in particular genes involved in drug metabolism. Second, a suitable model can be developed to determine hosts' responses to drugs before clinical treatment.

Antibiotic resistome in a large urban-lake drinking water source in middle China: Dissemination mechanisms and risk assessment.

The increasing pollution of urban drinking water sources by antibiotic resistance genes (ARGs) threatens human health worldwide. However, the distribution and influencing factors of ARGs, especially how to reveal the risks of ARGs in this environment remains unclear. Hence, Chaohu Lake was selected as an example to investigate the characteristics of ARGs and explore the interactions among physicochemical factors, microorganisms, and ARGs by metagenomic approach. In this work, 75 ARG subtypes with an average of 30.4 × /Gb (ranging from 15.2 ×/Gb to 57.9 ×/Gb) were identified, and multidrug and bacA were most frequent in Chaohu Lake. Non-random co-occurrence patterns and potential host bacteria of ARGs were revealed through co-occurrence networks. Microbial community and mobile genetic elements (MGEs) were the major direct factors in ARG profiles. The dissemination of ARGs was mainly driven by plasmids. Considering the interactions among MGEs, human bacterial pathogens, and ARGs, antibiotic resistome risk index (ARRI) was proposed to manifest the risks of ARGs. Overall, our work systemically investigated the composition and associated factors of ARGs and built ARRI to estimate the potential risks of ARGs in a typical urban drinking water source, providing an intuitive indicator for managing similar lakes.