Interactions between temperature and energy supply drive microbial communities in hydrothermal sediment.

Temperature and bioavailable energy control the distribution of life on Earth, and interact with each other due to the dependency of biological energy requirements on temperature. Here we analyze how temperature-energy interactions structure sediment microbial communities in two hydrothermally active areas of Guaymas Basin. Sites from one area experience advective input of thermogenically produced electron donors by seepage from deeper layers, whereas sites from the other area are diffusion-dominated and electron donor-depleted. In both locations, Archaea dominate at temperatures >45 °C and Bacteria at temperatures <10 °C. Yet, at the phylum level and below, there are clear differences. Hot seep sites have high proportions of typical hydrothermal vent and hot spring taxa. By contrast, high-temperature sites without seepage harbor mainly novel taxa belonging to phyla that are widespread in cold subseafloor sediment. Our results suggest that in hydrothermal sediments temperature determines domain-level dominance, whereas temperature-energy interactions structure microbial communities at the phylum-level and below.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity.

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.