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Methane occurred in the monimolimnion, at depths greater than 11 m, of an antarctic meromictic lake, Ace Lake (depth 24.7 m). Although the water of the lake was of approximate marine salinity, bottom waters were depleted in sulfate (less than 1 mmol l-'). The temperature of the bottom waters of the lake were constantly between 1 "C and 2 "C. Rates of methanogenesis from 14C-labelled precursors (bicarbonate, formate and acetate) were determined in time course experiments with the detection of 14CH4 produced by a gas chromatography-gas proportional counting system. Rates of 14CH4 production were difficult to determine as the reactions were always near our limit of detection.Reliable determinations of rates of methanogenesis at some depths using some precursors were obtained, the fastest rate being 2.5 pmol kg-' day-' at depth 20 m. Assuming constant rates of methanogenesis with time, this would equate to a turnover of methane in the lake every two years.The slow rate of methanogenesis suggests that the methanogens in Ace Lake may be working at well below their optimum temperature although definitive statements regarding the presence of psychrophilic methanogens in this antarctic lake must await isolation attempts or longer field studies using alternative methodologies.

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ∼36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.