Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

Integrin-specific control of focal adhesion kinase and RhoA regulates membrane protrusion and invasion.

Cell invasion through extracellular matrix (ECM) is a hallmark of the metastatic cascade. Cancer cells require adhesion to surrounding tissues for efficient migration to occur, which is mediated through the integrin family of receptors. Alterations in expression levels of ß1 and ß3 integrins have previously been reported in a number of human cancers. However, whether there are specific roles for these ubiquitous receptors in mediating cell invasion remains unclear. Here we demonstrate that loss of ß1 but not ß3 integrins leads to increased spread cell area and focal adhesion number in cells on 2D immobilized fibronectin. Increased adhesion numbers in ß1 knockdown cells correlated with decreased cell migration on 2D surfaces. Conversely, cells depleted of ß1 integrins showed increased migration speed on 3D cell-derived matrix as well as in 3D organotypic cultures and inverted invasion assays. This increased invasive potential was also seen in cells lacking ß3 integrin but only in 3D cultures containing fibroblasts. Mechanistically, in situ analysis using FRET biosensors revealed that enhanced invasion in cells lacking ß1 integrins was directly coupled with reduced activation of focal adhesion kinase (FAK) and the small GTPase RhoA resulting in formation of enhanced dynamic protrusions and increased invasion. These reductions in FAK-RhoA signal activation were not detected in ß3 knockdown cells under the same conditions. This data demonstrates a specific role for ß1 integrins in the modulation of a FAK-RhoA-actomyosin signaling axis to regulate cell invasion through complex ECM environments.