CRISPR screens identify gene targets at breast cancer risk loci.

BACKGROUND: Genome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in non-coding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS. RESULTS: Here, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants. CONCLUSIONS: We demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.

Simultaneous quantification of humic acid-water and silanized glass-water partition constants for PCBs, PCDDs and OCDF.

Super-hydrophobic organic contaminants (SHOCs) such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and octachlorodibenzofuran (OCDF) can sorb to dissolved hydrophobic materials including humic acids (HAs), enhancing their apparent aqueous solubility and potentially resulting in increased groundwater contamination and offsite transport. To manage risks associated with transport of and contamination by SHOCs, modelling approaches incorporating partitioning data, i.e. dissolved organic carbon-water partition constants (KDOC), are necessary. Measurement of KDOC can however be compromised by SHOC sorption to glassware surfaces leading to an overestimation of experimental values resulting in larger KDOC. A method for simultaneous derivation of KDOC and glass-water partition constants (KGW) is described. It involves a mass balance approach combined with HA as a co-solvent at various concentrations and accounts for SHOC losses to silanized glassware. Measured log KDOC values ranged from 5.28 to 7.64 for tetra- to decachlorinated PCBs, 6.67 to 7.93 for tetra- to octachlorinated PCDDs and 8.20 for OCDF. These data were linear functions of log KOW and consistent with relationships reported for more polar compounds. Log KGW (mm3 mm-2) values (1.62 to 4.06 for PCBs, 2.96 to 3.90 for PCDDs, 3.77 for OCDF) were one order of magnitude greater compared to literature PCB borosilicate glass-water partition constants. Techniques such as those presented in this work present simple, versatile means to provide prediction of the SHOC proportion remaining in aqueous solutions after loss to glassware that was inversely related to container surface area/volume ratio and log KOW in our study.

Cold Induced Antisense Transcription of FLOWERING LOCUS C in Distant Grasses.

Functional conservation of RNAs between different species is a key argument for their importance. While few long non-coding RNAs are conserved at the sequence level, many long non-coding RNAs have been identified that only share a position relative to other genes. It remains largely unknown whether and how these lncRNAs are conserved beyond their position. In Arabidopsis thaliana, the lncRNA COOLAIR is transcribed antisense from FLOWERING LOCUS C (FLC) in response to cold. Despite relatively low sequence similarity, the COOLAIR expression pattern and in vitro RNA secondary structure are highly conserved across the family Brassicaceae, which originated some 50 mya. It is unclear, however, whether COOLAIR functions in distantly related species such as monocots, which diverged some 150 mya. Here, we identified antisense lncRNAs from FLC homologs in various monocot species that share no sequence similarity with A. thaliana COOLAIR. Yet similar to COOLAIR, we found that BdODDSOC1 antisense (BdCOOLAIR1) and BdODDSOC2 antisense (BdCOOLAIR2) are induced by cold in a Brachypodium distachyon winter accession. Across B. distachyon accessions, the sequences of BdCOOLAIR1 and BdCOOLAIR2 are less conserved than exons but more conserved than flanking regions, suggesting a function for the transcript itself. Knock down of the BdODDSOC2 non-overlapping BdCOOLAIR2 transcript did not show a morphological phenotype, but did result in significantly higher BdODDSOC2 expression during cold, indicating that BdCOOLAIR2 performs a role in cis in the rate of BdODDSOC2 silencing. This functional similarity between eudicot and monocot species reveals ancient conservation or convergent evolution of FLC antisense transcription. Either scenario supports its functional importance.