Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets

Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening

Abstract Background Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control. Results We designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings. Conclusions In this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox

CRISPR/Cas9-targeting of CD40 in hematopoietic stem cells limits immune activation mediated by anti-CD40.

Inflammatory bowel diseases (IBD) are complex, multifactorial disorders characterized by chronic relapsing intestinal inflammation. IBD is diagnosed around 1 in 1000 individuals in Western countries with globally increasing incident rates. Association studies have identified hundreds of genes that are linked to IBD and potentially regulate its pathology. The further dissection of the genetic network underlining IBD pathogenesis and pathophysiology is hindered by the limited capacity to functionally characterize each genetic association, including generating knockout animal models for every associated gene. Cutting-edge CRISPR/Cas9-based technology may transform the field of IBD research by efficiently and effectively introducing genetic alterations. In the present study, we used CRISPR/Cas9-based technologies to genetically modify hematopoietic stem cells. Through cell sorting and bone marrow transplantation, we established a system to knock out target gene expression by over 90% in the immune system of reconstituted animals. Using a CD40-mediated colitis model, we further validated our CRISPR/Cas9-based platform for investigating gene function in experimental IBD. In doing so, we developed a model system that delivers genetically modified mice in a manner much faster than conventional methodology, significantly reducing the time from target identification to in vivo target validation and expediting drug development

A library of TAL effector nucleases spanning the human genome

Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-kappa B signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

Data showing positive regulation of Wnt/β-catenin signal transduction by FAM129B siRNA using transcriptional reporter assay, target gene expression, and apoptosis assay

<p>Data for Figure 2b. Measured β-catenin dependent firefly: constitutive renilla ratios for HT1080, A375, and A2058 reporter cells treated with the indicated conditions. Data for Figure 2c: Quantitative RT-PCR of β-catenin dependent Axin2 mRNA and housekeeping actin mRNA for HT1080, A375, and A2058 cells treated with the indicated conditions. Data for Figure 3a. Measured β-catenin dependent firefly: constitutive renilla ratios for A375 reporter cells treated with a dose curve of WNT3A or a single dose of WNT3A. Data for Figure 3b. Measured NFκB dependent firefly: constitutive renilla ratios for A375 reporter cells treated with a dose curve of TNFalpha or a single dose of TNFα . Data for Figure 4b. Quantitation of DAPI and TUNEL positive nuclei from A375 cells treated with WNT3A, PLX4720, and the control, FAM129B, or CTNNB1 siRNA.</p

UPDATED: Data sets and query script used in identifying FAM129B as a putative regulator of Wnt/β-catenin signaling using a largescale siRNA screen integrated with phosphoproteomic and bioinformatic analyses; and, FAM129B protein-protein interaction data.

<p>Results of affinity purification of Flag-GFP-FAM129B followed by mass spectrometric peptide identification. Column a, bait protein used for affinity purification. Column b, prey proteins identified by mass spectrometry. Column c, total peptides identified for a given prey peptide (spectral counts).</p

Data sets and query script used in identifying FAM129B as a putative regulator of Wnt/β-catenin signaling using large-scale siRNA screen integrated with phosphoproteomic and bioinformatic analyses.

<p>Data File 1. Results of a large-scale siRNA screen for Wnt/β-catenin signaling in HT1080 B/R cells. Column a contains the siRNA probe ID. Column b contains the ENTREZ gene ID. Coulnm D shows median percent enhancement or inhibition of BAR reporter relative to control siRNA. All data are normalized to constitutively expressed renilla. P-value calculated using student's T. if, for a given gene, all siRNA shows a twofold change in med(normFFluc) and a -logP <=2, that gene is classified as a hit. Data File 2. Melanoma-associated genes identified by an NCBI gene search. Data File 3a. Protein target overlaps between the siRNA screen and the phosphoproteomic dataset. Column A contains the official gene symbol for protein target of associated peptide. If, for a given gene, all siRNA shows a twofold change in med(normFFluc) and a -logP <=2, that gene is classified as a hit. Phosphoproteomic Hit defined as a 1.7-fold change in phosphosite intensity as described in Old WM et al. 2009. Gene previously associated with melanoma as identified by biopython code described in the Query Script. % control siRNA = median percent enhancement or inhibition of BAR reporter relative to control siRNA. All data are normalized to a viability marker. Data File 3b. Protein target overlaps between siRNA dataset and the bioinformatic enrichment. If, for a given gene, all siRNA shows a twofold change in med(normFFluc) and a -logP <=2, that gene is classified as a hit. median percent enhancement or inhibition of BAR reporter relative to control siRNA. All data are normalized to constitutively expressed renilla. Query Script. Biopython script for identifying disease- or query-associated geneIDs.</p

Small Interfering RNA Screens Reveal Enhanced Cisplatin Cytotoxicity in Tumor Cells Having both BRCA Network and TP53 Disruptions

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/−) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity ∼4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone