RNA Engineering for Public Health: Innovations in RNA-Based Diagnostics and Therapeutics

Abstract

RNA is essential for cellular function: From sensing intra- and extracellular signals to controlling gene expression, RNA mediates a diverse and expansive list of molecular processes. A long-standing goal of synthetic biology has been to develop RNA engineering principles that can be used to harness and reprogram these RNA-mediated processes to engineer biological systems to solve pressing global challenges. Recent advances in the field of RNA engineering are bringing this to fruition, enabling the creation of RNA-based tools to combat some of the most urgent public health crises. Specifically, new diagnostics using engineered RNAs are able to detect both pathogens and chemicals while generating an easily detectable fluorescent signal as an indicator. New classes of vaccines and therapeutics are also using engineered RNAs to target a wide range of genetic and pathogenic diseases. Here, we discuss the recent breakthroughs in RNA engineering enabling these innovations and examine how advances in RNA design promise to accelerate the impact of engineered RNA systems.

Keywords: CRISPR; RNA; diagnostic; public health; synthetic biology; therapeutic.

FIGURE 1

TIMELINE OF SELECT MILESTONES IN RNA ENGINEERING BUILDING TOWARD APPLICATIONS IN DIAGNOSTICS AND THERAPEUTICS. ABBREVIATIONS: DETECTR, DNA ENDONUCLEASE-TARGETED CRISPR TRANS REPORTER; FDA, US FOOD AND DRUG ADMINISTRATION; MRNA, MESSENGER RNA; SARS-COV-2, SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2; SHERLOCK, SPECIFIC HIGH-SENSITIVITY ENZYMATIC REPORTER UNLOCKING.

Figure 2

Summary of RNA-based pathogen biosensors. (a) General overview of RNA-based molecular diagnostics for pathogens. (b) Toehold switches control translation to modulate signal generation. They have been integrated into a paper-based sensor for Ebola detection. Panel adapted with permission from Pardee et al. (15). (c) STARs leverage transcriptional regulation to detect pathogenic nucleic acids. They have been used in a colorimetric assay to detect plant viruses. Panel adapted with permission from Verosloff et al. (https://pubs.acs.org/doi/10.1021/acssynbio.8b00526). Further permissions should be directed to the American Chemical Society. (33). (d) SHERLOCK and (e) DETECTR leverage collateral cleavage activity of Cas proteins to detect viral RNA. Panels adapted with permission from (d) Zhang et al. (36) and (e) Broughton et al. (37). SHERLOCK and DETECTR have been used to detect SARS-CoV-2. Abbreviations: crRNA, CRISPR RNA [**AU: Spell out?**]; DETECTR, DNA endonuclease-targeted CRISPR trans reporter; gRNA, guide RNA; PAM, protospacer adjacent motif [**AU: Spell out?**]; RBS, ribosome binding site [**AU: Spell out?**]; RT, reverse transcriptase; SHERLOCK, specific high-sensitivity enzymatic reporter unlocking; ssRNA, single-stranded RNA[**AU: Correct?** Yes]; STARS, small transcription activating RNA.

FIGURE 3

RNA CAN SERVE AS BOTH A SENSOR AND A REPORTER. (A) SCHEMATIC FOR DETECTION OF AQUEOUS FLUORIDE VIA THE CRCB FLUORIDE RIBOSWITCH. THE FLUORIDE APTAMER IN THE CRCB RIBOSWITCH BINDS FLUORIDE, WITH LIGAND BINDING CHANGING THE RNA SWITCH’S FINAL CONFORMATION TO ALLOW PRODUCTION OF A DOWNSTREAM ENZYMATIC REPORTER. THE ENZYME CONVERTS A COLORLESS COMPOUND INTO A YELLOW PIGMENT. WITHOUT FLUORIDE, TRANSCRIPTION OF THE REPORTER GENE IS PROHIBITED. PANEL ADAPTED WITH PERMISSION FROM THAVARAJAH ET AL. (HTTPS://PUBS.ACS.ORG/DOI/FULL/10.1021/ACSSYNBIO.9B00347). FURTHER PERMISSIONS SHOULD BE DIRECTED TO THE AMERICAN CHEMICAL SOCIETY. (17). (B) SCHEMATIC FOR ROSALIND (RNA OUTPUT SENSORS ACTIVATED BY LIGAND INDUCTION). CONTAMINANTS ARE DETECTED VIA A CONTAMINANT-SPECIFIC TRANSCRIPTION FACTOR, WITH LIGAND BINDING ENABLING THE PRODUCTION OF A FLUORESCENT RNA APTAMER. IN THE ABSENCE OF THE TARGET COMPOUND, TRANSCRIPTION IS BLOCKED. PANEL ADAPTED WITH PERMISSION FROM JUNG ET AL. (18).

FIGURE 4

ENHANCEMENT OF CAS9 EDITING RATE AND SPECIFICITY VIA GUIDE RNA (GRNA) ENGINEERING. (A) SCHEMATIC OF CAS9 EDITING MECHANISM. CAS9 MEDIATES THE SEQUENCE-SPECIFIC BINDING OF ITS GRNA SPACER SEQUENCE TO A CORRESPONDING SITE ON THE TARGET DNA. RECOGNITION INITIATES SITE-SPECIFIC DNA CLEAVAGE. CELLULAR RECOMBINATION MACHINERY THEN USES EXTERNALLY SUPPLIED SYNTHETIC DNA CONTAINING HOMOLOGY REGIONS TO THE DESIRED EDITING SITE TO REPAIR THE BREAK, ADDING THE DESIRED DONOR SEQUENCE TO THE DNA.[**AU: EDIT OK?** YES] (B) EXAMPLE GRNA MODIFICATIONS (BOLDED) AND FUNCTIONS. ENGINEERING THE GRNA WITH ADDITIONAL SEQUENCES AND CHEMICAL MODIFICATIONS CAN INCREASE CLEAVAGE EFFICIENCY AND GRNA STABILITY, REDUCE OFF-TARGET EDITING, AND CONFER NOVEL FUNCTIONS. (C) SCHEMATIC OF PRIME EDITING MECHANISM. AN RNA-GUIDED NICKASE/REVERSE TRANSCRIPTASE (RT) FUSION MEDIATES THE WRITING OF AN EDIT ENCODED IN THE GRNA. CELLULAR RECOMBINATION MACHINERY THEN SEALS THE NICK, INCORPORATING THE EDIT INTO THE GENOME.

FIGURE 5

ENGINEERING RNA SEQUENCES, STRUCTURES AND INTERACTIONS, FOR THERAPEUTIC PURPOSES. (A) SCHEMATIC OF AN SAM VACCINE BEING DELIVERED INTO THE CELL VIA VESICLE-BASED DELIVERY, RELEASED IN THE CELL, AMPLIFIED WITH VIRAL REPLICASE MACHINERY, TRANSLATED, AND EXPRESSED TO TRIGGER AN IMMUNE REACTION. (B) SCHEMATIC OF HOW AN ASO CAN BLOCK THE SPLICING MACHINERY TO PREVENT EXON REMOVAL AND THE RESULTING DISEASE CAUSED BY AN UNDERLYING MISSENSE MUTATION. (C) SCHEMATIC OF RNAI TARGETING TO A MRNA 3′ UTR, WHICH REDUCES PROTEIN PRODUCTION THROUGH MRNA DEGRADATION IF PERFECT BASE PAIRING OR TRANSLATIONAL DELAY IF MISMATCHED BASE PAIRING. (D) TWO MECHANISMS OF SMIRNAS THAT EITHER BLOCK RNA PROCESSES OR INDUCE RNA DEGRADATION. ABBREVIATIONS: SAM, SELF-AMPLIFIED MRNA; ASO, ANTISENSE OLIGONUCLEOTIDES; MRNA, MESSENGER RNA; ORF, OPEN READING FRAME; RNAI, RNA INTERFERENCE; SA, SELF-AMPLIFYING [**AU: SPELL OUT**]; SMIRNAS, SMALL MOLECULES INTERACTING WITH RNA; UTR, UNTRANSLATED REGION.