RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Sistemas CRISPR-Cas , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Células A549 , Profilaxis Antibiótica/métodos , Secuencia de Bases , Betacoronavirus/genética , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Simulación por Computador , Secuencia Conservada , Coronavirus/efectos de los fármacos , Coronavirus/genética , Coronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/tratamiento farmacológico , Proteínas de la Nucleocápside de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , Células Epiteliales/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Pulmón/patología , Pulmón/virología , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , Filogenia , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2 , Proteínas no Estructurales Virales/genéticaRESUMEN
A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Humanos , Liposomas , Nanopartículas , SARS-CoV-2/genéticaRESUMEN
CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.