F5 - CRISPR-Functionalized 3D Printed Platform for Spatiotemporal Control of Gene Editing

Introduction: : The clinical translation of clustered regularly interspaced short palindromic repeats (CRISPR) is hindered by its limited efficiency in achieving therapeutic levels of genome editing at the target site in vivo. Systemic administration of CRISPR components results in rapid degradation by proteases and nucleases in plasma, clearance from circulation, and accumulation in the liver, significantly reducing editing efficiency. Additionally, systemic injection can lead to toxicity and unexpected off-target effects in other tissues. As an alternative to systemic delivery, local injection of CRISPR/Cas9 has also been investigated to decrease systemic toxicity, showing reduced off-target DNA damage. However, local delivery is also hindered by low gene editing efficiency (0.3%-5.4%) due the dense extracellular matrix (ECM) which limits biodistribution, requiring repeated administration. Further, these bolus injections risk nanoparticle accumulation, toxicity, and inflammation. To address these limitations, in situ biomaterial-mediated delivery ensures the even distribution of CRISPR machinery, localizes editing to the injury site, and can extend the duration of nanoparticle release. However, current surface-adsorption strategies offer limited control over CRISPR loading, release kinetics, and spatial patterning. To overcome these challenges, our objective was to engineer a CRISPR-functionalized 3D-printed platform that enables precise spatiotemporal control of gene editing.

Materials and

Methods: : We first identified a peptide-based binding system that could be used to covalently functionalize cell penetrating peptide (CPP)-CRISPR nanoparticles to a 3D printed scaffold. The CPP-CRISPR nanoparticles were produced using the arginine-alanine-leucine-alanine (RALA) peptide. The snooptag system is inspired from an interdomain isopeptide bond in the pilus adhesin (RrgA) protein from Streptococcus pneumoniae. In this system, snooptag (KLGSIEFIKVNK) can form covalent bonds directly with the snoopcatcher protein or with the dogtag peptide (DIPATYEFTDGKHYITNEPIPPKGGGC) via snoopligase catalysis. The incorporation of snooptag on the surface of CPP-CRISPR nanoparticles allowed for nanoparticle binding to the dogtag or snoopcatcher molecules. Dogtag was then incorporated into 3D printed scaffolds using previously established solvent cast techniques that results in peptides evenly distributed on the surface of 3D printed poly(caprolactone) (PCL) scaffolds. To attach the snoopcatcher protein to the PCL scaffold, snoopcatcher was fused to the spycatcher protein which binds a spytag peptide (AHIVMVDAYKPTKGGGC) in a similar manner to the snooptag-snoopcatcher bond. This system therefore allowed the 3D printing of spytag and dogtag coated filaments at high resolution to enable the binding of unique CRISPR nanoparticles in defined patterns for spatial control over gene editing. The release kinetics of the CPP-CRISPR nanoparticles were analyzed by fluorescently labelling of the CRISPR cargos. The editing efficiency of the released nanoparticles was characterized using reporter mesenchymal stem cells (MSCs) harvested from the bone marrow of CAG-luc-eGFP L2G85 transgenic mice.

Results, Conclusions, and Discussions:: First, we confirmed effective CPP-CRISPR nanoparticle formation by characterizing the size and charge of the complexes upon addition of snooptag-RALA. Nanoparticles maintained diameters below 150 nm across all tested molar ratios of snooptag-RALA. However, when snooptag-RALA exceeded 20% of the formulation, a significant reduction in surface charge was observed, which correlated with reduced transfection efficiency in mesenchymal stem cells (MSCs), likely due to insufficient cationic charge to promote endocytosis. After optimizing nanoparticle formation and confirming efficient transfection, we next assessed whether snooptag peptides were properly presented on the nanoparticle surface for binding to snoopcatcher or dogtag moieties. To evaluate this, nanoparticles were reacted with either spytag-GFP or dogtag-GFP, and binding was quantified. The incorporation of snoopligase did not enhance binding compared to the dogtag-GFP control (NC), suggesting that observed interactions were primarily due to non-specific electrostatic forces between snooptag and spytag. In contrast, a significant increase in binding was observed in the snoopcatcher–spycatcher system compared to the spytag-GFP control (p < 0.0001), indicating effective, specific peptide-mediated interactions. Following confirmation of specific CPP-CRISPR nanoparticle binding to spytag and dogtag, we functionalized polycaprolactone (PCL) filaments with these peptide tags using a previously established solvent-cast 3D printing method. Fluorescently labeled CPP-CRISPR nanoparticles functionalized with snooptag were successfully tethered to either spytag- or dogtag-functionalized scaffolds, enabling sustained release over one week. To evaluate genome editing from these CRISPR-functionalized 3D printed scaffolds, we employed a transwell system in which the scaffold was placed in the upper insert and mouse MSCs expressing GFP and luciferase were cultured in the lower chamber. Flow cytometry and luciferase assays revealed prolonged silencing of both reporters, demonstrating sustained gene editing activity from the scaffold-bound CRISPR system. In conclusion, we established a modular 3D-printed platform enabling spatiotemporal control of CRISPR delivery through peptide-mediated nanoparticle tethering and sustained release, supporting its potential for localized genome editing. Future studies will explore the in vivo editing capacity of this approach for the correction of genetic diseases affecting the musculoskeletal system such as muscle dystrophy and osteogenesis imperfecta.

Acknowledgements and/or References (Optional)::