Cycle 1: Delivery Efficiency and Sustained Release

  • Acknowledge (The New Problem): Initial LNP formulations showed burst release profiles of the EYFP protein, which may not sustain CRISPRa expression throughout the critical antiviral window, reducing the efficacy of the system.
  • Research (The Exchange): Drawing from ZJU’s hydrogel system and controlled release strategy, we explored the use of thermo-responsive Poloxamer 407 hydrogels for extended LNP release.
  • Effect (The Impact): We developed a Hydrogel-LNP Matrix (HLNP) that encapsulates both plasmid DNA and sgRNAs, enabling gradual release over 72 hours and improving transfection stability in vitro.
  • Adjust (The Refinement): The HLNP system now includes DOTAP/Chol LNPs (3:1 molar ratio) with a controlled size of ~100 nm, complexed within a Poloxamer 407 hydrogel to facilitate co-culture delivery and extended intracellular activity.

Cycle 2: Gene Target Selection and Safety

  • Acknowledge (The New Problem): Initial gene targets, which included TLRs, were selected for antiviral potency, but their potential for inflammatory or off-target effects remained unaddressed.
  • Research (The Exchange): Learning from ZJU’s critiques on targeting downstream safety and non-inflammatory effectors, we revisited our gene selection criteria to prioritize targets with minimal immune activation.
  • Effect (The Impact): We incorporated RAB7A more prominently due to its role in viral degradation without triggering cytokine release, and added IFITM3, as the IFIT family of proteins effectively targets the many instances of membrane fusion, be it at entry, or during escape from late endosomes.
  • Adjust (The Refinement): Our sgRNA library now includes targets with 2′-O-methyl modifications at both ends to enhance stability and reduce immunogenicity, and we plan to validate each target using cytokine profiling assays.

Cycle 3: System Controllability and Safety

  • Acknowledge (The New Problem): The original CRISPRa system posed risks of leaky expression and potential long-term activation, without a clear indicator of viral specificty.
  • Research (The Exchange): Adopting concepts from ZJU’s dual-input suicide switch and feedback-regulated amplifiers, we decided to fuse our system with a TetON mechanism to limit prolonged CRISPRa activity.
  • Effect (The Impact): We introduced a Dox-dependent OFF-switch, using rtTA for TRE3G promoter activation, thereby adding a layer of delivery control. No Dox means no rtTA, means no CRISPRa.
  • Adjust (The Refinement): The revised system now includes rtTA under a constitutive EF1α promoter, allowing it to be produced, but remain inactive until Dox induction, at which point it can bind to the TRE3G promoter of dCas9 and start the CRISPRa system.

Conclusion

Through structured reflection and inspired by the ZJU iGEM team’s innovative work, we have systematically enhanced our CRISPRa-based antiviral platform across four key dimensions: context-aware activation, sustained delivery, refined targeting, and integrated safety. These improvements not only strengthen the efficacy and practicality of our system but also align it with emerging standards in synthetic biology for real-world therapeutic applications.