Biology subjects

McManus, M. T.

Publications and source records attributed to McManus, M. T..

3 recordsLinked to original sources

bioRxiv2017

Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding

While pooled loss- and gain-of-function screening approaches have become increasingly popular to systematically investigate mammalian gene function, they have thus far ignored the fact that cell populations are heterogeneous. Here we introduce multi-level barcoded sgRNA libraries to (i) monitor differences in the behavior of multiplexed clonal cell lines, (ii) trace sub-clonal lineages of cells expressing the same sgRNA, (iii) derive in-sample screen replicates and (iv) reduce the number of cells and sequencing read counts required to reach statistical significance. Using our approach, we illustrate how clonal heterogeneity impairs the results of pooled genetic screens and demonstrate the ability of multi-level barcoding to resolve cellular heterogeneity related issues.

genomics↗

bioRxiv2017

The transcriptionally permissive chromatin state of ES cells is acutely tuned totranslational output

A permissive chromatin environment coupled to hypertranscription is critical to drive the rapid proliferation of Embryonic Stem (ES) cells and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ES cells. The results reveal that activity of cellular growth pathways, prominently protein synthesis, perpetuates the euchromatic state and hypertranscription of ES cells. Acute, mild inhibition of translation results in rapid depletion of euchromatic marks in ES cells and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Remarkably, reduced translational output leads to rewiring of open chromatin within 3 hours, including decreased accessibility at a subset of active developmental enhancers and increased accessibility at histone genes and transposable elements. Using a proteome-scale analysis, we show that several euchromatin regulators are unstable proteins and thus continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription and translation sets the pace of proliferation at peri-implantation and may be employed generally by stem/progenitor cells.

molecular biology↗

bioRxiv2017

Decoding directional genetic dependencies through orthogonal CRISPR/Cas screens

Genetic interaction studies are a powerful approach to identify functional interactions between genes. This approach can reveal networks of regulatory hubs and connect uncharacterised genes to well-studied pathways. However, this approach has previously been limited to simple gene inactivation studies. Here, we present an orthogonal CRISPR/Cas-mediated genetic interaction approach that allows the systematic activation of one gene while simultaneously knocking out a second gene in the same cell. We have developed this concept into a quantitative and scalable combinatorial screening platform that allows the parallel interrogation of hundreds of thousands of genetic interactions. We demonstrate that the established platform works robustly to uncover genetic interactions in human cancer cells and to interpret the direction of the flow of genetic information.

molecular biology↗