TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.National Institutes of Health (U.S.) (Grant RO1-GM34277)National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706)Damon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramSimons Foundatio

Population structure, growth and production of the wedge clam Donax hanleyanus (Bivalvia: Donacidae) from northern Argentinean beaches

Donax hanleyanus Philippi, 1847 (Bivalvia: Donacidae) dominates fine to coarse sandy beach communities of the northern Argentinean Atlantic coast. The population biology of this intertidal wedge clam was studied by determining population structure, growth and production at the three locations Santa Teresita, Mar de las Pampas (both from December 2005 to December 2006) and Faro QuerandıŽ (from March 2005 to December 2006). Von Bertalanffy growth functions were established from length-frequency distributions using an asymptotic length (LN) of 44 mm and the growth constants (K) of 0.46 and 0.47 y1 respectively of Mar de las Pampas and Faro QuerandıŽ. Compared with growth studies four decades ago, D. hanleyanus today is growing more slowly, but is reaching a higher maximum length. Longevity is estimated to be approximately five years. The present study confirms that the overall growth performance index is habitat-specific, grouping Donacidae into tropical/subtropical, temperate and upwelling species. The intertidal biomass of D. hanleyanus ranged between 0.04 and 1.32 g ash-free dry mass (AFDM) m2yr1. Individual production revealed the highest value at 30 mm length (0.16 g AFDM m2yr1) and annual production ranged between 0.08 and 0.99 g AFDMm2yr1, resulting in renewal rate values (P/B) between 0.82 and 2.16. The P/B ratios of D. hanleyanus populations increased with decreasing latitude from temperate to tropical regions. Only at Santa Teresita D. hanleyanus was found living with the sympatric yellow clam Mesodesma mactroides. A significant negative correlation between abundances of both surf clams suggests that abundance peaks of D. hanleyanus are related with population crashes of M. mactroides. Spatial differences in abundance are significantly related to sand texture as confirmed by nonmetrical multidimensional scaling, but not to sea surface temperature. However, the decrease of D. hanleyanus seems to be principally related to human activities

Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.National Institutes of Health (U.S.) (Grant HD 045022)National Institutes of Health (U.S.) (Grant R37CA084198

Selfish, sharing and scavenging bacteria in the Atlantic Ocean: a biogeographical study of bacterial substrate utilisation

Identifying the roles played by individual heterotrophic bacteria in the degradation of high molecular weight (HMW) substrates is critical to understanding the constraints on carbon cycling in the ocean. At five sites in the Atlantic Ocean, we investigated the processing of organic matter by tracking changes in microbial community composition as HMW polysaccharides were enzymatically hydrolysed over time. During this investigation, we discovered that a considerable fraction of heterotrophic bacteria uses a newly-identified ‘selfish’ mode of substrate processing. We therefore additionally examined the balance of individual substrate utilisation mechanisms at different locations by linking individual microorganisms to distinct substrate utilisation mechanisms. Through FISH and uptake of fluorescently-labelled polysaccharides, ‘selfish’ organisms were identified as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. ‘Sharing’ (extracellular enzyme producing) and ‘scavenging’ (non-enzyme producing) organisms predominantly belonged to the Alteromonadaceae and SAR11 clades, respectively. The extent to which individual mechanisms prevail depended on the initial population structure of the bacterial community at a given location and time, as well as the growth rate of specific bacteria. Furthermore, the same substrate was processed in different ways by different members of a pelagic microbial community, pointing to significant follow-on effects for carbon cycling