King Abdullah University of Science and Technology (KAUST) 7 articles published in JoVE Biology Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV Atheer Althobaiti1, Kareem Hamdan1, Mohamed A. Sobhy1, Renad Rawas1, Masateru Takahashi1, Olga Artyukh1, Muhammad Tehseen1 1Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST) We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory. Genetics Chromatin Immunoprecipitation in the Cnidarian Model System Exaiptasia diaphana Mascha Fiona Dix*1, Peng Liu*2, Guoxin Cui1, Francesco Della Valle2, Valerio Orlando2, Manuel Aranda1 1Red Sea Research Center (RSRC), Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST), 2KAUST Environmental Epigenetics Program (KEEP), Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST) A chromatin immunoprecipitation (X-ChIP) assay against the histone mark H3K4me3 for the model organism Exaiptasia diaphana is presented. The specificity and effectiveness of the assay are confirmed by quantitative PCR and next-generation sequencing. This protocol enables the increased investigation of protein-DNA interactions in the sea anemone E. diaphana. Immunology and Infection Gene Editing of Primary Rhesus Macaque B Cells Harald Hartweger1, Rajeev Gautam2, Yoshiaki Nishimura2, Fabian Schmidt3,5, Kai-Hui Yao1, Amelia Escolano1,6, Mila Jankovic1, Malcolm A. Martin2, Michel C. Nussenzweig1,4 1Laboratory of Molecular Immunology, The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Retrovirology, The Rockefeller University, 4Howard Hughes Medical Institute, The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center, Wistar Institute We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies. Biology Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use Pedro M. Cardoso1, Ahmed A. Alsaggaf1, Helena M. Villela1, Raquel S. Peixoto1 1Red Sea Research Center (RSRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST) Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments. Engineering Proof-of-Concept for Gas-Entrapping Membranes Derived from Water-Loving SiO2/Si/SiO2 Wafers for Green Desalination Ratul Das1, Sankara Arunachalam1, Zain Ahmad1, Edelberto Manalastas1, Ahad Syed2, Ulrich Buttner2, Himanshu Mishra1 1Water Desalination and Reuse Center (WDRC), Biological and Environmental Science and Engineering (BESE) Division, King Abdullah University of Science and Technology (KAUST), 2Core Labs, King Abdullah University of Science and Technology (KAUST) Presented here is a stepwise protocol for realizing gas-entrapping membranes (GEMs) from SiO2/Si wafers using integrated circuit microfabrication technology. When silica-GEMs are immersed in water, the intrusion of water is prevented, despite the water-loving composition of silica. Engineering Rendering SiO2/Si Surfaces Omniphobic by Carving Gas-Entrapping Microtextures Comprising Reentrant and Doubly Reentrant Cavities or Pillars Sankara Arunachalam1, Eddy M. Domingues1, Ratul Das1, Jamilya Nauruzbayeva1, Ulrich Buttner2, Ahad Syed2, Himanshu Mishra1 1Water Desalination and Reuse Center (WDRC), Biological and Environmental Science and Engineering (BESE) Division, King Abdullah University of Science and Technology (KAUST), 2Core Labs, King Abdullah University of Science and Technology (KAUST) This work presents microfabrication protocols for achieving cavities and pillars with reentrant and doubly reentrant profiles on SiO2/Si wafers using photolithography and dry etching. Resulting microtextured surfaces demonstrate remarkable liquid repellence, characterized by robust long-term entrapment of air under wetting liquids, despite the intrinsic wettability of silica. Genetics Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1 Mohamed A. Sobhy*1, Amer Bralić*1, Vlad-Stefan Raducanu1, Muhammad Tehseen1, Yujing Ouyang1, Masateru Takahashi1, Fahad Rashid1, Manal S. Zaher1, Samir M. Hamdan1 1Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology (KAUST) Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.
