Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015–2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies.

Zooplankton dominate the abundance and biomass of multicellular animals in pelagic marine environments; however, traditional methods to characterize zooplankton communities are invasive and laborious. This study compares zooplankton taxonomic composition revealed through metabarcoding of the cytochrome oxidase I (COI) and 18S rRNA genes to traditional morphological identification by microscopy. Triplicates of three different sample types were collected from three coral reef sites in the Florida Keys National Marine Sanctuary: (1) 1 L surface seawater samples prefiltered through 3 μm filters and subsequently collected on 0.22 μm filters for eDNA (PF‐eDNA); (2) 1 L surface seawater samples filtered on 0.22 μm pore‐size filters (environmental DNA; eDNA), and (3) zooplankton tissue samples from 64 μm, 200 μm, and 500 μm mesh size net tows. The zooplankton tissue samples were split, with half identified morphologically and tissue DNA (T‐DNA) extracted from the other half. The COI and 18S rRNA gene metabarcoding of PF‐eDNA, eDNA, and T‐DNA samples was performed using Illumina MiSeq. Of the families detected with COI and 18S rRNA gene metabarcoding, 40% and 32%, respectively, were also identified through morphological assessments. Significant differences in taxonomic composition were observed between PF‐DNA, eDNA, and T‐DNA with both genetic markers. PF‐eDNA resulted in detection of fewer taxa than the other two sample types; thus, prefiltering is not recommended. All dominant copepod taxa (> 5% of total abundance) were detected with eDNA, T‐DNA, and morphological assessments, demonstrating that eDNA metabarcoding is a promising technique for future biodiversity assessments of pelagic zooplankton in marine systems.

Environmental DNA (eDNA) is an emerging and powerful method for use in marine research, conservation, and management, yet time‐ and resource‐intensive protocols limit the scale of implementation. Long‐range autonomous underwater vehicles equipped with autonomous environmental sample processors (LRAUV‐ESPs) provide a new means for scaling up marine eDNA sample collection and processing. Here, we used eDNA metabarcoding of four marker genes (mitochondrial 12S rRNA, bacterial and archaeal 16S rRNA, nuclear 18S rRNA, and mitochondrial COI), which encompass the diversity of marine species from microbes to vertebrates, to demonstrate the efficacy of an LRAUV‐ESP in sampling eDNA and assessing community structure in the Monterey Bay National Marine Sanctuary. The sequencing results from samples that were autonomously collected were comparable with those collected from a ship at similar locations, times, and depths, supporting previous results that found no significant differences using targeted qPCR. This study demonstrates the potential of equipping autonomous underwater vehicles with ESPs to greatly expand the scale of eDNA sample collection and processing and provide much needed information regarding the changing spatial and temporal patterns of marine biodiversity, especially in many data‐poor regions of the world's oceans.

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