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Jun 26, 2023 414 likes •558 views

Genome-Wide CRISPR-Cas9 Screening for Identification of Cancer Essential Genes in Malignant Pleural Mesothelioma Ece akrolu, MSc. Izmir Biomedicine and Genome Center & Dokuz Eylul University, Izmir International Biomedicine and

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Genome-Wide CRISPR-Cas9 Screening for Identification of Cancer Essential Genes in Malignant Pleural Mesothelioma

Izmir Biomedicine and Genome Center & Dokuz Eylul University, Izmir International Biomedicine and Genome Institute ece.cakiroglu@msfr.ibg.edu.tr

Ece Çakıroğlu, MSc.

Funded by TUBITAK, project no: 117Z227

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Malignant Pleural Mesothelioma

➢ 80-90% of mesothelioma cases ➢ Asbestos exposure

Irritation
Oxygen free radicals
Interfering mitosis
Inflammatory cytokines
Phosphorylation of protein kinases

➢ Rare ➢ Increasing incidance ➢ Late diagnosis ➢ Invasive character ➢ Poor survival

Bibby AC, Tsim S et al., Eur Respir Rev 2016 Kato S, Tomson BN et al., Mol Cancer Ther 2016

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)

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SLIDE 3

Our Aim

Identification and Functional Characterization of Essential Genes Involved in the Development of Malignant Pleural Mesothelioma

Whole- Genome CRISPR Screen

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SLIDE 4

Non-homologous end joining Homology directed repair

Jinek M, Chylinski K et al., Science 2012 Ran FA et al., Nature Protocols 2013 28.10.2019 4

SLIDE 5

CRISPR Screens

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Clonal and Stable Cas9 Expression

Transfection Infection Sorting

HEK293T

Cell Line 96-well plates seeded Obtained Clones

MeT-5A 9 12 H2052 15 13 H2452 6 12 H28 10 11 28.10.2019 6

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Choosing The Rig

ight Clo lone

MeT-5A H2052 H2452 H28

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Met-5A Met-5A Clone 1 Met-5A Clone 2 Met-5A Clone 3 Met-5A Clone 4 Met-5A Clone 5 Flag α-tubulin H2452 H2452 Clone 1 H2452 Clone 2 H2452 Clone 4 H2452 Clone 6 H2452 Clone 9 H28 H28 Clone 11 H28 Clone 2 H28 Clone 4 H28 Clone 6 H28 Clone 5 H2052 H2052 Clone 13 H2052 Clone 4 H2052 Clone 5 H2052 Clone 12 H2052 Clone 8 Flag α-tubulin Flag α-tubulin Flag α-tubulin

Choosing The Rig

ight Clo lone

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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

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SLIDE 10

Addgene Senturk Perfectly Matching Guides (%) 93.5 93.6 Undetected Guides (%) 0.2 0.3 Skew Ratio 4.50 4.46

Lentiviral gRNA Library Amplification

NGS Result

LentiGuide-Puro plasmid backbone

76441 sgRNs Brunello gRNA Library Target sequence sgRNA

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Large-Scale Virus Production

Brunello : psPAX2 : pMD2.G 4 : 2 : 1 DNA : PEI 1 : 5

MOI Calculation

MeT-5A ➔ 1/128 H28 ➔ 1/128 H2052 ➔ 1/48 H2452 ➔ 1/24

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Sample collection T=0 T=7 T=14 NGS MOI Calculation

Bioinformatical Analysis Determining candidates Validation

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THANK YOU FOR YOUR ATTENTION

Senturk Lab Members

－Özlem Şilan Coşkun －Aslı Kurden Pekmezci －Dilara Demirci －Bengisu Uluata Dayanç －F. Aybüke Mazı

Advisor

－Doç Dr. Şerif Şentürk

Funded by TUBITAK, project no: 117Z227

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