for Identification of Cancer Essential Genes in Malignant Pleural - PowerPoint PPT Presentation

Genome-Wide CRISPR-Cas9 Screening for Identification of Cancer Essential Genes in Malignant Pleural Mesothelioma Ece Çakıroğlu, MSc. Izmir Biomedicine and Genome Center & Dokuz Eylul University, Izmir International Biomedicine and Genome Institute ece.cakiroglu@msfr.ibg.edu.tr Funded by TUBITAK, project no: 117Z227

Malignant Pleural Mesothelioma ➢ 80-90% of mesothelioma cases ➢ Asbestos exposure - Irritation ➢ Late diagnosis - Oxygen free radicals ➢ Invasive character - Interfering mitosis ➢ Poor survival - Inflammatory cytokines - Phosphorylation of protein kinases ➢ Rare ➢ Increasing incidance • Multimodal/trimodal treatment ➔ standard in the treatment of early-stage MPM • Poor prognosis • Low survival (~1-year mean survival; 9% 5-year survival ) • Pemetrexed - a folic acid-like chemotherapeutic agent- (FDA approval in 2004) Bibby AC, Tsim S et al., Eur Respir Rev 2016 28.10.2019 2 Kato S, Tomson BN et al., Mol Cancer Ther 2016

Our Aim Identification and Functional Characterization of Essential Genes Involved in the Development of Malignant Pleural Mesothelioma Whole- Genome CRISPR Screen 28.10.2019 3

Homology directed repair Non-homologous end joining Jinek M, Chylinski K et al., Science 2012 28.10.2019 4 Ran FA et al., Nature Protocols 2013

CRISPR Screens 28.10.2019 5

Clonal and Stable Cas9 Expression Infection Transfection Sorting HEK293T 96-well plates Obtained Cell Line seeded Clones MeT-5A 9 12 H2052 15 13 H2452 6 12 H28 10 11 28.10.2019 6

Choosing The Rig ight Clo lone MeT-5A H2052 H2452 H28 28.10.2019 7

Choosing The Rig ight Clo lone Met-5A Met-5A Met-5A Met-5A H2052 H2052 H2052 H2052 H2052 H2052 Met-5A Met-5A Clone 2 Clone 3 Clone 4 Clone 5 Clone 4 Clone 12 Clone 13 Clone 1 Clone 5 Clone 8 Flag Flag α -tubulin α -tubulin H28 H28 H28 H28 H28 H2452 H2452 H2452 H2452 H2452 H2452 H28 Clone 2 Clone 4 Clone 6 Clone 11 Clone 1 Clone 2 Clone 4 Clone 6 Clone 9 Clone 5 Flag Flag α -tubulin α -tubulin 28.10.2019 8

Testing Functionality of Cas9 Competition Assay MeT-5A-Cas9 H2052-Cas9 lentiGuide-Hygro-dTomato-Ren g.208 H2452-Cas9 lentiGuide-Hygro-dTomato-RPA3 g.44 H28-Cas9 28.10.2019 9

Lentiviral gRNA Library Amplification 76441 sgRNs plasmid backbone LentiGuide-Puro Target sequence sgRNA Brunello gRNA Library NGS Result Addgene Senturk Perfectly Matching 93.5 93.6 Guides (%) Undetected 0.2 0.3 Guides (%) Skew Ratio 4.50 4.46 28.10.2019 10

Large-Scale Virus Production Brunello : psPAX2 : pMD2.G 4 : 2 : 1 DNA : PEI 1 : 5 MeT-5A ➔ 1/128 H2052 ➔ 1/48 MOI Calculation H2452 ➔ 1/24 H28 ➔ 1/128 28.10.2019 11

Bioinformatical Analysis MOI Determining Calculation candidates Validation NGS T=0 T=7 T=14 Sample collection 28.10.2019 12

THANK YOU FOR YOUR ATTENTION • Advisor － Doç Dr. Şerif Şentürk • Senturk Lab Members － Özlem Şilan Coşkun － Aslı Kurden Pekmezci － Dilara Demirci － Bengisu Uluata Dayanç － F. Aybüke Mazı Funded by TUBITAK, project no: 117Z227 28.10.2019 13