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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I10 - Optimizing CRISPR-Cas9 RNP Delivery with the DIV3W Peptide: Addressing Cellular Uptake and Brand Variability

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Gene editing is a powerful tool for disease treatments and diagnostic tools, with the potential to transform the medical industry. One gene editing tool is clustered regularly interspaced short palindromic repeats associated with Cas genes, CRISPR-Cas9. The CRISPR-Cas9 system consists of a single-stranded guide RNA (sgRNA) guiding a DNA nuclease Cas9 to a gene. Cas9 can be delivered in many forms, including plasmid DNA, mRNA, or ribonucleoprotein (RNP). RNP Cas9 has higher editing efficiency and reduced off-target effects, avoiding the instability and integration issues found in plasmid and mRNA Cas9. However, there remain many barriers for clinical applications of RNP CRISPR-Cas9, including cellular uptake, endosomal escape, and nuclear entry. To overcome these barriers, peptides have emerged as a promising delivery method. Peptides form through self-assembly, can escape the endosome and respond to stimuli, have a low immunogenic response, and are highly customizable based on cell type and intended function. One promising peptide is DIV3W, a fusogenic peptide that facilitates endosomal escape and RNA complexation through electrostatic interactions. This study investigates DIV3W as an industry equivalent delivery vehicle for CRISPR-Cas9 RNP and compares the impact of Cas9 RNP brand on treatment uptake in healthy human embryonic kidney (HEK293) cells. By addressing these critical delivery problems and Cas9 source variability in gene editing drug delivery, this research aims to advance practical applications of CRISPR-Cas9 and the clinical potential of gene editing therapeutics.

Materials and

Methods:  CRISPR-Cas9 RNP complexes were formed through self-assembly by mixing equimolar concentrations of CRISPR-Cas9 from two different brands with gRNA or non-targeting siRNA (siNT). Cas9 RNP was sourced from Integrated DNA Technologies, Inc. (IDT, Coralville, IL) or Varizymes (Middleton, WI). gRNA was used to evaluate knockout efficiency when combined with CRISPR-Cas9 RNP, subsequently termed gRNP. siNT evaluated treatment internalization, and termed siRNP. Treatment groups were formed by adding DIV3W at varying molar ratios to the complexes. Impact of the Cas9 brands was first evaluated through dynamic light scattering (DLS), of free Cas9, siRNP, gRNP, and gRNP:DIV3W peptides at a 1:40 molar ratio. Next, RNP:peptide complexation was evaluated using gel shifts run at 100V for 45 mins in a 2% agarose gel. Banding was visualized with ethidium bromide staining.

For internalization studies, RNP:DIV3W treatments with Cy5 siRNA were added to healthy HEK293-GFP cells using Lipofectamine as a positive control. The uptake efficiency of both Varizyme and IDT Cas9s was qualitatively and quantitatively assessed through fluorescence microscopy and flow cytometry 4-hrs post-treatment.

Results, Conclusions, and Discussions: Both IDT and Varizyme Cas9 RNPs demonstrated interaction with both RNA and the DIV3W peptide, as observed by size changes in DLS (Fig. 1). Notably, IDT gRNP:DIV3W complexes exhibited a particle size decrease of over 300 nm to 169 nm, while Varizyme gRNP:DIV3W experienced particle size decreases of over 650 nm to 129 nm. Similarly, complexation with the positively charged DIV3W also caused the particles to have a net positive charge. These results confirm peptide conjugation through charge-based compaction and zeta potential shifts from negative towards positive. The positive charge and smaller particle size seen in the DIV3W treatments are advantageous to cellular interaction and uptake, aiding in overcoming delivery barriers for gene editing.

Gel-shift assays confirmed successful binding of gRNA by both Cas9 types, demonstrated by the lack of free gRNA banding at the bottom of the gel (Fig. 2). However, Varizyme’s Cas9 has incomplete binding to siNT, as shown by the bright white band at the bottom similar to free siNT. Even though both Cas9s are isolated from S. pyogenes, a considerable difference exists between brands in complexation with RNA cargo, highlighting key differences in product manufacturing.

Flow cytometry quantified HEK293 cellular uptake after 4 hrs of treatment, using Lipofectamine as the industry-standard positive control. Both Varizyme and IDT RNP:DIV3W treatments had increased uptake when compared to Lipofectamine, both in quantity of events (Fig. 3A) and signal intensity (Fig. 3B). Varizyme Cas9 seemingly had the best uptake results, due to DIV3W binding to the unbound siRNA (Fig. 2) during RNP:DIV3W complexation. This is important because DIV3W:siRNA complexes only provide transient gene alterations, and the lack of Cas9 in the complex prevents any long-term therapeutic benefits.

These findings demonstrate the potential of peptides to enhance CRISPR-Cas9 delivery and overcome key barriers such as efficient cellular uptake. The differences between Cas9 brands underscore the importance of material sourcing when working with CRISPR-Cas9, as different brands may interact differently with drug delivery cargo/vehicles. This work highlights the therapeutic potential of fusogenic peptides like DIV3W in improving gene editing efficiency for disease treatment, paving the way for advancements in CRISPR-Cas9 clinical applications.

Acknowledgements (Optional):  This work is supported by the Clemson Creative Inquiry Program, NSF EPSCoRprogram, NSF Award #OIA-1655740, NSF Faculty Early Career Development Program, NSF Award #204669, and Clemson CECAS Undergraduate Grant #211.