This week’s interesting science

Nature: Common genetic variation drives molecular heterogeneity in human iPSCs (Kilpinen et al.). The first results form the large-scale HipSci initiative funded by Wellcome.

A very interesting Nature study on vascular development and how it is regulated by FGF-dependent control of metabolism (glycolysis) via Myc: FGF-dependent metabolic control of vascular development (Yu et al. 2017). Here is a copy of the final conclusion of the paper: The FGF/MYC/HK2-dependent regulation of vascular development is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic16 and in the systemic vasculature17, injury response18 and maintenance of endothelium-to-mesenchymal transition both in the lymphatic and in the systemic vasculature, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors and in the systemic vasculature17, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors involved, which of the 22 FGF family members is responsible for the required FGF signalling input is not known. In summary, FGF signalling regulates blood and lymphatic vascular development through control of endothelial metabolism driven by MYC-dependent regula-tion of HK2 expression. Therapeutic targeting of this FGF–MYC–HK2 pathway may open new possibilities for treatment of diseases associated with insufficient or excessive vascular growth.

Just out in Nature Medicine: Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients (Zehir et al. 2017) ➡ Interesting recurrence of certain well-known oncogenes (e.g. PIK3CA) across a range of tumour types, irrespective of lineage, but also very interesting to see how mutations in certain oncogenes are absent from specific tumour histotypes. TP53 mutations top the list and correlate positively with more aggressive cancers. Most of the TP53 mutations were inactivating due to truncation or altered splicing. Second on the list is KRAS G12 followed by PIK3CA H1047R and E545K.

Cell Metabolism: PPARδ Promotes Running Endurance by Preserving Glucose (Fan et al. 2017). Just skimmed through, looks quite comprehensive. PPARd orchestrates the transcriptional switch to fat utilisation and glucose sparing in response to exercise.

Cell Metabolism: DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging (Park et al. 2017) – a mechanism that involves phosphorylation of Hsp90 and inhibition of AMPK.

An interesting paper in Cell (Stem Cell Lineage Infidelity Drives Wound Repair and Cancer by Ge et al.) that uncovers how tumour cells hijack normal wound repair processes which under normal circumstances allow for transient stem cell lineage infidelity. In contrast, a pre-cancerous stem cells are locked into this plastic state, giving rise to excessive growth and ultimately full-blown cancer.

Nature Protocols: Assessment of engineered cells using CellNet and RNA-seq (Radley et al. 2007) – a how-to-guide for the computational platform CellNet, which allows one to upload one RNAseq data from a particular cell type / stage and compare it to large datasets on different cell types. This should be very useful for estimation of cell fate transitions in response to different differentiation perturbations.

 

eLife: Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering (Lee et al. 2017). These guys test different modifications of sgRNAs and ssODN repair templates and show that they can be tolerated. For instance, they tag the ssODN with an Alexa-647 which allows them to enrich for cells that have been successfully transfected with the repair template. They also manage to fuse the sgRNA and ssODN template to each other, complex these with Cas9 (RNP), and show that this improved cellular delivery based on cationic polymers. However, note that despite this improval the efficiency of gene editing is still lower compared to nucleofection/electroporation under comparable conditions.

Nature Methods: CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets (Tsai et al. 2017). A new genome-wide method is described for the assessment of CRISPR off-target effects in vitro and following tag integration into host cell DNA. Much more sensitive compared to previous methods that achieved whole-genome assessment of off-target effects. A second study in Nature Methods reports on a different method (SITE-seq) that also aims to profile genome-wide off-target effects with high sensitivity: Mapping the genomic landscape of CRISPR–Cas9 cleavage (Cameron et al. 2017). However, the specificity of Cas9-mediated gene editing in a cell will also be highly cell-specific and depend  on the concentration of enzyme and additional components that are delivered for gene editing (sgRNA, ssODN etc.). The effect of increasing sgRNA and Cas9 concentrations in vitro is assessed in the biochemical assay reported by Cameron et al. Both of the above methods achieve a much higher sensitivity of off-target site detection because they specifically enriched for DNA fragments associated with Cas9 cleavage prior to sequencing (i.e. all the random background is removed). Once such off-targets are identified genome-wide, they can be interrogated in any cell line that has been edited – many will, however, remain intact in the cell because of the effects of chromatin on target accessibility as well as cell-specific DNA repair mechanisms.

 

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