CRISPR screens identify candidate host factors for SARS and MERS coronavirus infection

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that can cause severe respiratory disease in humans. Identification of the host factors that are necessary for viral infection and virus-induced cell death is critical to our understanding of the viral life cycle and can potentially aid the development of new treatment options. Here, we report CRISPR screen results of both SARS-CoV and MERS-CoV infections in derivatives of the human hepatoma cell line Huh7. Our screens identified the known entry receptors ACE2 for SARS-CoV and DPP4 for MERS-CoV. Additionally, the SARS-CoV screen uncovered several components of the NF-?B signaling pathway (CARD10, BCL10, MALT1, MAP3K7, IKBKG), while the MERS-CoV screen revealed the polypyrimidine tract-binding protein PTBP1, the ER scramblase TMEM41B, furin protease and several transcriptional and chromatin regulators as candidate factors for viral replication and/or virus-induced cell death. Together, we present several known and unknown coronavirus host factors that are of interest for further investigation. Overall design: All experiments were performed under required biosafety conditions. For the SARS-CoV screen, a genome-wide knockout gene library using the human GeCKOv2 library (Addgene, #1000000049, gift from Feng Zhang) (PMID: 25075903) was generated in clonal Huh7.5.1-Cas9 cells expressing ACE2-IRES-TMPRSS2 (PMID: 33333024). 144 million cells were infected with SARS-CoV at an MOI of 0.0001. Surviving cells were harvested at 12 days post-infection in RLT buffer and genomic DNA was extracted with AllPrep DNA/RNA spin columns (Qiagen). Eluted DNA was irradiated for biosafety reasons. Library cells at day 0 were extracted as baseline control. For the MERS-CoV screen, Huh7.5.1-Cas9 cells transduced with the human GeCKOv2 library were infected with MERS-CoV at an MOI of 0.0001. The furin inhibitor Decanoyl-RVKR-CMK was added to the media at 5 µM throughout the experiment to prevent excessive syncytia formation. Surviving cells were harvested in DNA/RNA Shield (Zymo) at 8 days post-infection and genomic DNA was extracted using the Quick-DNA Midiprep Plus Kit (Zymo). Uninfected, Decanoyl-RVKR-CMK-treated library cells were cultured in parallel and harvested for comparison. Library cells at day 0 were extracted as baseline control. The sgRNA expression cassettes were amplified from genomic DNA in a two-step nested PCR using KAPA HiFi HotStart ReadyMixPCR Kit (Kapa Biosystems) and sequenced on an Illumina NextSeq 500 using a custom sequencing primer, as previously described (PMID: 33333024). Demultiplexed FASTQ files were processed using MaGeCK (PMID: 25476604). First, FASTQ files were aligned to a reference table containing sgRNA sequences to determine the counts of each sgRNA in each control and selected cell population. Then, count tables for GeCKO sublibraries A and B were combined and gene enrichment was calculated from sgRNA count tables using the total norm-method.