Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by VLP

With sequencing as a standard frontline protocol to identify emerging viruses such Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), direct utilization of sequence data to program antivirals against the viruses could accelerate drug development to treat their infections. CRISPR-Cas effectors are promising candidates that could be programmed to inactivate viral genetic material based on sequence data, but several challenges such as delivery and design of effective CRISPR RNA (crRNA) need to be addressed to realize practical use. Here, we showed that virus-like particle (VLP) could deliver PspCas13b-crRNA ribonucleoprotein (RNP) in nanomolar range to efficiently suppress dengue virus infection in primary human target cells. Shortening spacer length could significantly enhance RNA-targeting efficiency of PspCas13b in mammalian cells compared to the natural length of 30 nucleotides without compromising multiplex targeting by a crRNA array. Our results demonstrate the potentials of applying PspCas13b RNP to suppress RNA virus infection, with implications in targeting host RNA as well. [Display omitted] We construct a virus-like particle that can efficiently deliver Cas13b-crRNA ribonucleoprotein complex into primary human cells to inhibit dengue virus infection. In addition, short spacer length of crRNA (22–26 nucleotides) can enhance Cas13b knockdown activity in mammalian cells without interfering with crRNA processing and enabling multi-virus targeting.