RESUMO
Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/genética , Mapeamento Cromossômico , Epistasia Genética/genética , Genoma Humano/genética , Células HEK293 , Humanos , RNA de Transferência/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Right ventricular failure (RVF) remains the leading cause of death in pulmonary arterial hypertension (PAH). We investigated the transcriptomic signature of RVF in hemodynamically well-phenotyped monocrotaline (MCT)-treated, male, Sprague-Dawley rats with severe PAH and decompensated RVF (increased right ventricular (RV) end diastolic volume (EDV), decreased cardiac output (CO), tricuspid annular plane systolic excursion (TAPSE) and ventricular-arterial decoupling). RNA sequencing revealed 2547 differentially regulated transcripts in MCT-RVF RVs. Multiple enriched gene ontology (GO) terms converged on mitochondria/metabolism, fibrosis, inflammation, and angiogenesis. The mitochondrial transcriptomic pathway is the most affected in RVF, with 413 dysregulated genes. Downregulated genes included TFAM (-0.45-fold), suggesting impaired mitochondrial biogenesis, CYP2E1 (-3.8-fold), a monooxygenase which when downregulated increases oxidative stress, dehydrogenase/reductase 7C (DHRS7C) (-2.8-fold), consistent with excessive autonomic activation, and polypeptide N-acetyl-galactose-aminyl-transferase 13 (GALNT13), a known pulmonary hypertension (PH) biomarker (-2.7-fold). The most up-regulated gene encodes Periostin (POSTN; 4.5-fold), a matricellular protein relevant to fibrosis. Other dysregulated genes relevant to fibrosis include latent-transforming growth factor beta-binding protein 2 (LTBP2), thrombospondin4 (THBS4). We also identified one dysregulated gene relevant to all disordered transcriptomic pathways, ANNEXIN A1. This anti-inflammatory, phospholipid-binding mediator, is a putative target for therapy in RVF-PAH. Comparison of expression profiles in the MCT-RV with published microarray data from the RV of pulmonary artery-banded mice and humans with bone morphogenetic protein receptor type 2 (BMPR2)-mutations PAH reveals substantial conservation of gene dysregulation, which may facilitate clinical translation of preclinical therapeutic and biomarkers studies. Transcriptomics reveals the molecular fingerprint of RVF to be heavily characterized by mitochondrial dysfunction, fibrosis and inflammation.
