Repurposing CRISPR/Cas to Discover SARS‐CoV‐2 Detecting and Neutralizing Aptamers

Abstract

RNA aptamers provide useful biological probes and therapeutic agents. New methodologies to screen RNA aptamers will be valuable by complementing the traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Meanwhile, repurposing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated systems (Cas) has expanded their utility far beyond their native nuclease function. Here, CRISmers, a CRISPR/Cas-based novel screening system for RNA aptamers based on binding to a chosen protein of interest in a cellular context, is presented. Using CRISmers, aptamers are identified specifically targeting the receptor binding domain (RBD) of the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two aptamer leads enable sensitive detection and potent neutralization of SARS-CoV-2 Delta and Omicron variants in vitro. Intranasal administration of one aptamer, further modified with 2'-fluoro pyrimidines (2'-F), 2'-O-methyl purines (2'-O), and conjugation with both cholesterol and polyethylene glycol of 40 kDa (PEG40K), achieves effective prophylactic and therapeutic antiviral activity against live Omicron BA.2 variants in vivo. The study concludes by demonstrating the robustness, consistency, and potential broad utility of CRISmers using two newly identified aptamers but switching CRISPR, selection marker, and host species.

Aptamer-based assembly systems for SARS-CoV-2 detection and therapeutics

“…2). 72–74 Among them, bead-based SELEX with the substrates of magnetic beads, Ni-Sepharose, and Talon is currently the most common SELEX for screening virus-targeting aptamers due to its simple operation and time-saving properties. However, the immobilization of SARS-CoV-2 protein targets may result in a different conformation from their natural conformation and hide some of their epitopes.…”

“…192 The combination of double modifications, such as 2′-F and 2′-OMe, could further enhance the binding affinity. 72 As an isomer of 2′-F, FANA is the 2′-F substituent of arabino nucleic acid, and the derived aptamers that are obtained from a screening pool entirely composed of FANA sequences can resist degradation by RNase, while maintaining high-affinity with RBD. 193–195 PS modification is the first modification method for delivering nucleic acid in vivo and the most widely used.…”

“…Additionally, other modifications, such as cholesterol-conjugated PEG (polyethylene glycol) moiety, can enhance the inhibitory effect of aptamers on viruses by promoting cell membrane permeation and presentation. 72…”

Engineered aptamers for molecular imaging

“…65 However, the enzymatic fragility of RNA derives from the fact that most ribonucleases attack the phosphodiester bond by polarizing the 2′-hydroxyl group in the RNA aptamer. Consequently, the chemical substitution of the RNA 2′-hydroxyl group is an effective way to improve the bio-stability of RNA aptamers, such as 2′-fluoro group, 66 2′-amino, and 2′- O -methyl, 67–69 and locked nucleic acids (LNAs) 70 (Fig. 3B(a)).…”

Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement

“…POCT methods typically employ isothermal amplification to eliminate the need for complex thermal cycling procedures. , This includes techniques such as rolling circle amplification (RCA), − strand displacement amplification (SDA), , recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). − Direct staining of isothermally amplified products with dyes is sufficiently sensitive but may fall short on specificity due to the nonspecific nature of dye staining on nucleic acids. , Numerous reports have combined isothermal amplification with CRISPR/Cas to implement high sensitivity and specificity simultaneously by harvesting the sequence-specific capability of Cas endonuclease, which, however, necessitates the introduction of additional enzymes, increasing the cost and complexity. − …”