Abstract: Event Abstract Back to Event CRISPR/Cas9 mediated BFL-1 knock-out in nasopharyngeal carcinoma (NPC) cell lines Siti Fairus Abdul Rahman1*, Nethia Mohana-Kumaran1, Mohd Ghows Mohd Azzam1, Nur Adelina Mohd Norudin2 and Kalaivani Muniandy1 1 University of Science, Malaysia, Malaysia 2 Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM, Malaysia Background BCL-2 family proteins which are divided into pro- and anti-apoptotic proteins are critical regulators of the intrinsic apoptosis pathway. The anti-apoptotic proteins are upregulated in many cancers and have become attractive therapeutic targets. ABT-263, which inhibit anti-apoptotic proteins BCL-2, BCL-XL and BCL-w, has little effect on Nasopharyngeal carcinoma (NPC) cells, made it clear that other anti-apoptotic proteins such as MCL-1 or BFL-1/A1 are important in survival of NPC cells. Previous studies show that Epstein-Barr virus proteins LMP1 and EBV nuclear antigen 2 (EBNA2) increase the level of BFL-1 mRNA in Burkitts lymphoma cell lines, promoting its survival. Given that NPC is closely associated with EBV infection and most NPC patients in Malaysia are positive for EBV infection, we believe that BFL-1 together with other anti-apoptotic proteins could contribute to NPC survival in a collaborative manner. In this study, the functional role of BFL-1 is determined by knocking-out the gene using the CRISPR/Cas9 technology. The long-term goal of the study is to test the sensitivity of the BFL-1 knockout NPC cells to specific BCL-2 inhibitors to dissect the contribution of each of the anti-apoptotic proteins for NPC survival. Methods The BFL-1 gene was knocked-out in two NPC cell lines, HK1 and C666-1 cell line using the CRISPR/Cas9 genome editing tool. Results Two BFL-1 guide RNAs were designed and cloned into vector PX458 (pSpCas9(BB)-2A-GFP) and successful ligation was confirmed with PCR and Sanger sequencing. BFL-1 recombinant plasmids were transfected into NPC cell lines HK-1 and C666-1 using the TransIT-X2 Dynamic Delivery System from MirusTM. Single cell clones of the BFL-1 knockout NPC cells were generated through serial dilution. Conclusion To this end, targeting BFL-1 by CRISPR/Cas9 did not cause rapid cell death indicating that NPC cells do not solely depend on BFL-1 for survival and require inhibition of other anti-apoptotic proteins to mediate cell death. Acknowledgements This work is supported by the FRGS grant (203.PBIOLOGI.6711541), USM Short term grant (304/PBIOLOGI/6313312), and MAKNA Cancer Research Award (304/PBIOLOGI/650828/M121). We also would like to thank Molecular Pathology Unit, Institute for Medical Research, Kuala Lumpur for providing us with HK-1 and C666-1 cells after obtaining permission from Professor George Tsao (University of Hong Kong) for the HK-1 cells and Professor KW Lo (Chinese University of Hong Kong) for the C666-1 cells. Keywords: nasopharyngeal carcinoma, CRISPR/Cas9, BFL-1 (BCL2A1), BCL-2-related protein A1, Anti-apoptotic proteins, Apoptosis Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Abdul Rahman S, Mohana-Kumaran N, Mohd Azzam M, Mohd Norudin N and Muniandy K (2019). CRISPR/Cas9 mediated BFL-1 knock-out in nasopharyngeal carcinoma (NPC) cell lines. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00029 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Nov 2018; Published Online: 17 Jan 2019. * Correspondence: Mrs. Siti Fairus Abdul Rahman, University of Science, Malaysia, George Town, Malaysia, [email protected] Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Siti Fairus Abdul Rahman Nethia Mohana-Kumaran Mohd Ghows Mohd Azzam Nur Adelina Mohd Norudin Kalaivani Muniandy Google Siti Fairus Abdul Rahman Nethia Mohana-Kumaran Mohd Ghows Mohd Azzam Nur Adelina Mohd Norudin Kalaivani Muniandy Google Scholar Siti Fairus Abdul Rahman Nethia Mohana-Kumaran Mohd Ghows Mohd Azzam Nur Adelina Mohd Norudin Kalaivani Muniandy PubMed Siti Fairus Abdul Rahman Nethia Mohana-Kumaran Mohd Ghows Mohd Azzam Nur Adelina Mohd Norudin Kalaivani Muniandy Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.