CRISPR-Cas based strategies against HIV-1 and human coronaviruses

CRISPR-Cas systems offer versatile and powerful tools to combat persistent and emerging viral infections. This thesis investigates DNA- and RNA-targeting CRISPR strategies against HIV-1 and human coronaviruses to define design principles for antiviral therapeutics and diagnostics.
For HIV-1, we evaluated SaCas9 and CjCas9 in infected T-cell models. SaCas9 with a single gRNA efficiently eliminated all detectable wild-type proviruses. Selected dual-gRNA pairs achieved up to 97% excision of large proviral fragments, depending on rapid and kinetically compatible cleavage at both target sites. The compact CjCas9 also demonstrated potent HIV-1 inhibition: a single gRNA produced strong viral suppression, and dual-gRNA designs further improved antiviral activity, although rare proviral excision was observed. These findings highlight the importance of gRNA selection and nuclease choice in achieving effective viral inhibition, providing a framework for future gene-editing strategies aimed at a functional HIV cure.
To extend antiviral potential beyond DNA targeting, we explored RNA-targeting Cas13d to directly disrupt SARS-CoV-2 RNA replication. Cas13d was designed against highly conserved viral RNA sequences and effectively inhibited replication. Building on this, we developed a pan-coronavirus Cas13d strategy targeting a highly conserved region of the nsp12 gene, demonstrating broad activity against multiple human coronaviruses. The same crRNAs were adapted into a SHERLOCK-based diagnostic platform for sensitive and specific detection of human coronaviruses, demonstrating that CRISPR technologies can be applied for both therapeutic and diagnostic purposes.
Overall, this thesis defines key mechanistic and design principles—nuclease choice, gRNA design, cleavage kinetics, and delivery considerations—advancing CRISPR-based antiviral intervention and pandemic preparedness.