IW-Scoring: an Integrative Weighted Scoring framework for annotating and prioritizing genetic variations in the noncoding genome

Cancer Research UK Centre of Excellence Award to Barts Cancer Centre [C16420/A18066]; Barts and The London Charity Grant [MGU0344]; D.U. is funded by Pancreatic Cancer Research Fund Tissue Bank Grant. Funding for open access charge: Charity Open Access Fund (COAF) award

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We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.00

Contribution of Heritability and Epigenetic Factors to Skeletal Muscle Mass Variation in United Kingdom Twins

Context: Skeletal muscle mass (SMM) is one of the major components of human body composition, with deviations from normal values often leading to sarcopenia.Objective: Our major aim was to conduct a genome-wide DNA methylation study in an attempt to identify potential genomic regions associated with SMM.Design: This was a mixed cross-sectional and longitudinal study.Setting: Community-based study.Participants: A total of 1550 middle-aged UK twin (monozygotic and dizygotic) twins, 297 of which were repeatedly measured participated in the study.Main Outcome Measure: Appendicular lean mass assessed using DXA technology, and MeDIP-seq DNA methylation profiling genome-wide were obtained from each individual.Results: Heritability estimate of SMM, with simultaneous adjustment for covariates obtained using variance decomposition analysis was h2=0.809±0.050. After quality control and analysis of longitudinal stability, the DNA methylation data comprised of 723,029 genomic sites, with positive correlations between repeated measurements (Rrepeated =0.114–0.905). Correlations between MZ and DZ twins were 0.51 and 0.38 at a genome-wide average, respectively and clearly increased with Rrepeated. Testing for DNA methylation association with SMM in 50 discordant MZ twins revealed 36,081 nominally significant results, of which the top-ranked 134 signals (P&lt;0.01 and Rrepeated&gt;0.40) were subjected to replication in the sample of 1,196 individuals. Seven SMM-methylation association signals replicated at a false discovery rate &lt;0.1, and these were located in or near genes DNAH12, CAND1, CYP4F29P, ZFP64 which have previously been highlighted in muscle-related studies. Adjusting for age, smoking and blood cell heterogeneity did not alter significance of these associations.Conclusion: This epigenome-wide study, testing longitudinally stable methylation sites discovered and replicated a number of associations between DNA methylation at CpG loci and skeletal muscle mass. Four replicated signals were related to genes with potential muscle functions, suggesting that the methylome of whole blood may be informative of SMM variation<br/

Unravelling the molecular basis for regulatory T-cell plasticity and loss of function in disease

Regulatory T cells (Treg) are critical for preventing autoimmunity and curtailing responses of conventional effector T cells (Tconv). The reprogramming of T-cell fate and function to generate Treg requires switching on and off of key gene regulatory networks, which may be initiated by a subtle shift in expression levels of specific genes. This can be achieved by intermediary regulatory processes that include microRNA and long noncoding RNA-based regulation of gene expression. There are well-documented microRNA profiles in Treg and Tconv, and these can operate to either reinforce or reduce expression of a specific set of target genes, including FOXP3 itself. This type of feedforward/feedback regulatory loop is normally stable in the steady state, but can alter in response to local cues or genetic risk. This may go some way to explaining T-cell plasticity. In addition, in chronic inflammation or autoimmunity, altered Treg/Tconv function may be influenced by changes in enhancer-promoter interactions, which are highly cell type-specific. These interactions are impacted by genetic risk based on genome-wide association studies and may cause subtle alterations to the gene regulatory networks controlled by or controlling FOXP3 and its target genes. Recent insights into the 3D organisation of chromatin and the mapping of noncoding regulatory regions to the genes they control are shedding new light on the direct impact of genetic risk on T-cell function and susceptibility to inflammatory and autoimmune conditions.Timothy Sadlon Cheryl Y Brown Veronika Bandara Christopher M Hope John E Schjenken Stephen M Pederson James Breen Alistair Forrest Marc Beyer Sarah Robertson Simon C Barr