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

Ekapot Singsuksawat; Suppachoke Onnome; Pratsaneeyaporn Posiri; Amporn Suphatrakul; Nittaya Srisuk; Rapirat Nantachokchawapan; Hansa Praneechit; Chutimon Sae-Kow; Pala Chidpratum; Khanit Sa-Ngiamsuntorn; Suradej Hongeng; Panisadee Avirutnan; Thaneeya Duangchinda; Bunpote Siridechadilok

doi:10.1016/j.omtm.2021.04.014

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

Mol Ther Methods Clin Dev. 2021 Jun 11:21:729-740. doi: 10.1016/j.omtm.2021.04.014. Epub 2021 May 1.

Authors

Ekapot Singsuksawat¹, Suppachoke Onnome¹, Pratsaneeyaporn Posiri¹, Amporn Suphatrakul¹, Nittaya Srisuk¹, Rapirat Nantachokchawapan¹, Hansa Praneechit^{2

3}, Chutimon Sae-Kow¹, Pala Chidpratum¹, Khanit Sa-Ngiamsuntorn⁴, Suradej Hongeng⁵, Panisadee Avirutnan^{2

6}, Thaneeya Duangchinda¹, Bunpote Siridechadilok¹

Affiliations

¹ National Center for Genetic Engineering and Biotechnology, Klong Luang, Pathumthani 12120, Thailand.
² Division of Dengue Hemorrhagic Fever Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
³ Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
⁴ Department of Biochemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand.
⁵ Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand.
⁶ Siriraj Center of Research Excellence in Dengue and Emerging Pathogens, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

Abstract

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.

Keywords: CRISPR-Cas13; RNA virus; VLP; and virus-like particle; delivery; dengue; flavivirus; zika.