Project description:Generation of transgenic cell lines is limited by inefficient gene editing requiring genotypic screening of hundreds to thousands of colonies to isolate correctly gene-edited cells. Here, we describe a novel method called CRISPRa On-Target Editing Retrieval (CRaTER) that enriches for cells with on-target knock-in of a promoterless cDNA-fluorescent reporter transgene by transiently overexpressing the targeted endogenous genetic locus and sorting for fluorescent cells. We use CRaTER to enrich for rare cells with heterozygous, biallelic-editing of the endogenous, transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSC), resulting in a 9-fold enrichment compared to antibiotics selection alone. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNV) in MYH7, a gene encoding for sarcomeric MHC-β wherein autosomal dominant missense mutations cause cardiac and skeletal myopathies. CRaTER enabled 90% enrichment of heterozygous, biallelically-edited hiPSCs – a 38.6-fold enrichment compared to antibiotics selection alone – to generate 113 SNVs comprising 78 missense variants in MYH7. hiPSCs that have undergone CRaTER enrichment can differentiate to cardiomyocytes and exhibit expected localization and expression of MHC-β fusion proteins. Together, CRaTER substantially reduces the screening required for isolation of gene-edited cells, enabling the generation of transgenic cell lines at unprecedented scale.
Project description:Inheritance and plasticity of epigenetic divergence characterise early stages of speciation in an incipient cichlid species of an African crater lake.
Project description:This project aims to investigate the metabolic pathways expressed by the active microbial community occurring at the deep continental subsurface. Subsurface chemoLithoautotrophic Microbial Ecosystems (SLiMEs) under oligotrophic conditions are supported by H2; however, the overall ecological trophic structures of these communities are poorly understood. Some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa appear to support inverted trophic pyramids wherein methanogens contributing <5% of the total DNA apparently produce CH4 that supports the rest of the community. Here we show the active metabolic relationships of one such trophic structure by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Four autotrophic β-proteobacteria genera that are capable of oxidizing sulfur by denitrification dominate. They co-occur with sulfate reducers, anaerobic methane oxidizers and methanogens, which each comprises <5% of the total community. Defining trophic levels of microbial chemolithoautotrophs by the number of transfers from the initial abiotic H2-driven CO2 fixation, we propose a top-down cascade influence of the metabolic consumers that enhances the fitness of the metabolic producers to explain the inverted biomass pyramid of a multitrophic SLiME. Symbiotic partnerships are pivotal in the deep biosphere on and potentially beyond the Earth.
Project description:For the first time quantitative data on the abundance of Bacteria, Archaea, and Eukarya in deep terrestrial sediments are provided using multiple methods (total cell counting, quantitative real-time PCR, Q-PCR and catalyzed reporter deposition-fluorescence in situ hybridization, CARD-FISH). The oligotrophic (organic carbon content of ?0.2%) deep terrestrial sediments in the Chesapeake Bay area at Eyreville, Virginia, USA, were drilled and sampled up to a depth of 140?m in 2006. The possibility of contamination during drilling was checked using fluorescent microspheres. Total cell counts decreased from 10(9) to 10(6) cells/g dry weight within the uppermost 20?m, and did not further decrease with depth below. Within the top 7?m, a significant proportion of the total cell counts could be detected with CARD-FISH. The CARD-FISH numbers for Bacteria were about an order of magnitude higher than those for Archaea. The dominance of Bacteria over Archaea was confirmed by Q-PCR. The down core quantitative distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes as well as functional genes involved in different biogeochemical processes was revealed by Q-PCR for the uppermost 10?m and for 80-140?m depth. Eukarya and the Fe(III)- and Mn(IV)-reducing bacterial group Geobacteriaceae were almost exclusively found in the uppermost meter (arable soil), where reactive iron was detected in higher amounts. The bacterial candidate division JS-1 and the classes Anaerolineae and Caldilineae of the phylum Chloroflexi, highly abundant in marine sediments, were found up to the maximum sampling depth in high copy numbers at this terrestrial site as well. A similar high abundance of the functional gene cbbL encoding for the large subunit of RubisCO suggests that autotrophic microorganisms could be relevant in addition to heterotrophs. The functional gene aprA of sulfate reducing bacteria was found within distinct layers up to ca. 100?m depth in low copy numbers. The gene mcrA of methanogens was not detectable. Cloning and sequencing data of 16S rRNA genes revealed sequences of typical soil Bacteria. The closest relatives of the archaeal sequences were Archaea recovered from terrestrial and marine environments. Phylogenetic analysis of the Crenarchaeota and Euryarchaeota revealed new members of the uncultured South African Gold Mine Group, Deep Sea Hydrothermal Vent Euryarchaeotal Group 6, and Miscellaneous Crenarcheotic Group clusters.
Project description:Analyses of microbial diversity in marine sediments have identified a core set of taxa unique to the marine deep biosphere. Previous studies have suggested that these specialized communities are shaped by processes in the surface seabed, in particular that their assembly is associated with the transition from the bioturbated upper zone to the nonbioturbated zone below. To test this hypothesis, we performed a fine-scale analysis of the distribution and activity of microbial populations within the upper 50 cm of sediment from Aarhus Bay (Denmark). Sequencing and qPCR were combined to determine the depth distributions of bacterial and archaeal taxa (16S rRNA genes) and sulfate-reducing microorganisms (SRM) (dsrB gene). Mapping of radionuclides throughout the sediment revealed a region of intense bioturbation at 0-6 cm depth. The transition from bioturbated sediment to the subsurface below (7 cm depth) was marked by a shift from dominant surface populations to common deep biosphere taxa (e.g., Chloroflexi and Atribacteria). Changes in community composition occurred in parallel to drops in microbial activity and abundance caused by reduced energy availability below the mixed sediment surface. These results offer direct evidence for the hypothesis that deep subsurface microbial communities present in Aarhus Bay mainly assemble already centimeters below the sediment surface, below the bioturbation zone.
Project description:Probing the texture of the rare biosphere: We have bracketed the dimensions of the rare biosphere in two marine bacterial samples by comparing a deep (1 million sequences per sample) pyrosequencing analysis of the two samples and the cultures isolated from one of them.
Project description:Macrofauna is known to inhabit the top few 10s cm of marine sediments, with rare burrows up to two metres below the seabed. Here, we provide evidence from deep-water Permian strata for a previously unrecognised habitat up to at least 8 metres below the sediment-water interface. Infaunal organisms exploited networks of forcibly injected sand below the seabed, forming living traces and reworking sediment. This is the first record that shows sediment injections are responsible for hosting macrofaunal life metres below the contemporaneous seabed. In addition, given the widespread occurrence of thick sandy successions that accumulate in deep-water settings, macrofauna living in the deep biosphere are likely much more prevalent than considered previously. These findings should influence future sampling strategies to better constrain the depth range of infaunal animals living in modern deep-sea sands. One Sentence Summary: The living depth of infaunal macrofauna is shown to reach at least 8 metres in new habitats associated with sand injections.