Oral Presentation Melbourne Immunotherapy Network Winter Symposium 2021

CRISPR-Cas13b can potently suppress replication of SARS-CoV-2 and mutant strains (#15)

Mohamed Fareh 1 2 , Wei Zhao 3 , Wenxin Hu 1 2 , Joshua Casan 1 2 , Amit Kumar 2 3 , Jori Symons 3 , Ilia Voskoboinik 1 2 , Paul G Ekert 1 2 4 5 , Kanta Subbarao 6 7 , Damian Purcell 7 , Joseph A Trapani 1 2 , Sharon Lewin 3 8 9
  1. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, 3000, Australia
  2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, 3052, Australia
  3. Department of Infectious Diseases, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, 3000, Australia
  4. Murdoch Children’s Research Institute, Parkville, 3052, Australia
  5. Children’s Cancer Institute, Randwick, NSW, Australia
  6. WHO Collaborating Centre for Research and Reference in Influenza, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
  7. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, , Melbourne 3000, Australia
  8. Victorian Infectious Diseases Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
  9. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, 3010, Australia

Background:  There are currently no effective antiviral agents for COVID-19 and the development of novel SARS-CoV-2 antiviral drugs will likely take years. We hypothesised that CRISPR-Cas13b could effectively target and destroy SARS CoV2 RNA and could therefore be a novel strategy for the prevention and treatment of SARS-CoV-2 infection. This approach could also be used for future novel RNA virus.

Methods: We employed genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. HEK293T cells were co-transfected with plasmids expressing SARS-CoV-2 RNA with an eGFP reporter together with either pspCas13 gRNAs targeting the transcript of SARS-CoV-2 or a non-targeting (NT) gRNA as a control. gRNAs were linked to BFP to determine transfection efficiency. BFP and eGFP were quantified by fluorescence microscopy. Vero cells were transfected with gRNAs and after 24-72 hours were infected with two strains of SARS-CoV-2 virus including wild type or a D614G mutant. Inhibition of viral replication by gRNAs was examined in supernatant using either a 50% cell culture infectious dose (TCID50) assay or real-time qPCR for SARS-CoV-2 RNA.

Results: Reprogrammed Cas13b effectors targeting accessible regions of SARS-CoV2 Spike, non structural protein (NSP) or Nucleocapsid (NP) transcripts showed high specificity and achieved >98% silencing in a virus-free transfection model. Following infection with wild type SARS-CoV-2 in Vero cells, multiplexing gRNA targeting Spike, NSP or NP suppressed viral replication by up to 90% when measuring RNA or infectivity in culture supernatants (figure 1). Comprehensive mutagenesis of guide-target interactions demonstrated that mismatches longer than 9-nt at the 5’end completely abrogated silencing. Similarly, 3-nt mismatches placed internally (positions 14-16) or at the 3’ end were well tolerated, while 6-nt mismatches reduced silencing by ~30%. Single-nucleotide mismatches did not significantly affect degradation of RNA. Using infectious virus, gRNAs inhibited wild type and the D614G mutant with similar efficiency after adjusting for input viral inoculum.

Conclusion: Reprogrammed pspCas13b can act as a promising tool to silence SARS-CoV-2. The high specificity, efficiency and rapid deployment properties described here provide a molecular blueprint of anti-viral therapeutics that could prevent or potentially treat SARS-CoV2 as well as other new RNA viruses that may emerge in the future.