Photoactivatable CRISPR/Cas13d via upconversion nanoparticles for deep tissue RNA engineering and orthopedic therapy

Area of Science:

• Biotechnology
• Molecular Biology
• Biomedical Engineering

Background:

• Spatiotemporal control of RNA therapeutics is crucial for clinical applications but remains challenging.
• Existing methods often lack precision and deep-tissue penetration capabilities.

Purpose of the Study:

• To develop a non-invasive, light-controlled RNA manipulation system for deep tissues.
• To engineer a photoactivatable CRISPR/Cas13d (paCas13d) system for precise RNA targeting.
• To combine paCas13d with upconversion nanoparticles for near-infrared light activation.

Main Methods:

• Structure-guided engineering of RfxCas13d for light-switchable fragments using optogenetic dimerization (CRY2PHR/CIBN).
• Development of polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) for near-infrared to blue light conversion.
• In vivo application of the UCNPs-PEI@paCas13d system in a murine model of steroid-associated osteonecrosis.

Main Results:

• Achieved precise spatiotemporal RNA targeting within bone tissue using NIR-activated paCas13d.
• Demonstrated robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis driving glucocorticoid-induced osteocyte apoptosis.
• Prevented bone deterioration, preserving trabecular architecture and enhancing bone volume in treated mice.

Conclusions:

• The UCNPs-PEI@paCas13d platform offers non-invasive, optical control for RNA modulation in deep tissues.
• This system provides a versatile approach for treating diseases requiring localized RNA intervention with minimal systemic effects.
• Combines CRISPR/Cas13d programmability with optical control for advanced therapeutic strategies.