SnapShot: Insulin/IGF1 Signaling.

The insulin/IGF1signaling pathway (ISP) plays an essential role in long-term health. Some perturbations in this pathway are associated with diseases such as type 2 diabetes; other perturbations extend lifespan in worms, flies, and mice. The ISP regulates many biological processes, including energy storage, apoptosis, transcription, and cellular homeostasis. Such regulation involves precise rewiring of temporal events in protein phosphorylation networks. For an animated version of this Enhanced SnapShot, please visit http://www.cell.com/cell/enhanced/odonoghue.

Visual analytics of signalling pathways using time profiles.

Data visualisation is usually a crucial first step in analysing and exploring large-scale complex data. The visualisation of proteomics time-course data on post-translational modifications presents a particular challenge that is largely unmet by existing tools and methods. To this end, we present Minardo, a novel visualisation strategy tailored for such proteomics data, in which data layout is driven by both cellular topology and temporal order. In this work, we utilised the Minardo strategy to visualise a dataset showing phosphorylation events in response to insulin. We evaluated the visualisation together with experts in diabetes and obesity, which led to new insights into the insulin response pathway. Based on this success, we outline how this layout strategy could be automated into a web-based tool for visualising a broad range of proteomics time-course data. We also discuss how the approach could be extended to include protein 3D structure information, as well as higher dimensional data, such as a range of experimental conditions. We also discuss our entry of Minardo in the international DREAM8 competition.

Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.