Benchmarking LNP vs Electroporation for eGFP RNA and CRISPR-Cas9 Delivery in HSCs

BACKGROUND
Are LNPs a viable alternative to electroporation for CRISPR delivery in HSCs?

Electroporation has long been the standard method for CRISPR delivery in hematopoietic stem cells (HSCs), but its high efficiency can come with significant cytotoxicity which limits it to ex vivo use. Lipid nanoparticles (LNPs) are now emerging as a promising non-viral alternative, offering strong biocompatibility, improved cell viability, and greater potential for in vivo applications.

This study was carried out in two phases. First, LNPs produced using the TAMARA microfluidic platform were tested for mRNA delivery in HSCs using eGFP-mRNA as a model cargo. Second, LNP-mediated delivery was compared directly with electroporation in a CRISPR knockout assay targeting the β2-microglobulin (B2M) gene. Transfection efficiency, genome-editing activity, and post-transfection viability were measured to benchmark both approaches.

Results showed that LNPs can deliver RNA to HSCs with high efficiency and minimal cytotoxicity, while achieving gene-editing levels comparable to electroporation. Although further optimization may be needed depending on the payload and target cell type, LNPs represent a scalable, less invasive, and more translationally relevant approach to RNA-based genome editing in hematopoietic models.

INTRODUCTION
How Does Efficient Co-Delivery of Cas9 mRNA and sgRNA Improve CRISPR-Cas9 Gene Editing?

CRISPR–Cas9 technology has revolutionized genome engineering, enabling precise and programmable editing for therapeutic applications. Its use is expanding rapidly across fields such as immuno-oncology, hematology, and regenerative medicine. Discovered in the early 2000’s from bacterial adaptive immunity, the CRISPR–Cas9 system delivery has been improved using sgRNA to guide the Cas9 endonuclease to the targeted genomic sites. Later, the convergence of synthetic mRNA and lipid nanoparticle (LNP) technology has offered an elegant solution to reduce off-target activity by yielding transient Cas9 expression. Efficient co-delivery of Cas9 mRNA and sgRNA is thus critical to achieving robust and reproducible gene editing.

Hematopoietic stem cells (HSCs) are a major focus for gene therapy because of their essential role in regenerating the blood and immune systems. However, they are difficult to transfect due to their quiescent state and fragile membrane integrity, making the choice of delivery method critical to success.

Electroporation has long been a reference method for delivering RNA and CRISPR reagents into HSCs, but it is often associated with high cellular stress and reduced viability. Lipid nanoparticles (LNPs), by contrast, have emerged as a leading non-viral RNA delivery platform, offering scalability, tunable composition, and compatibility with both in vivo and ex vivo applications.

This study first used LNPs to deliver eGFP mRNA into HSCs to assess baseline transfection efficiency and cell viability across different RNA doses. It then evaluated the co-delivery of Cas9 mRNA and sgRNA in a CRISPR knockout model targeting β2-microglobulin (B2M), a cell-surface protein that can be readily measured by flow cytometry, to compare genome-editing performance.

LNPs were formulated using the TAMARA microfluidic system to support precise, reproducible, and scalable nanoparticle production at R&D scale. The aim of this study was to benchmark electroporation and LNP-mediated delivery by comparing transfection efficiency, editing activity, and post-transfection viability, helping researchers identify the most suitable RNA delivery strategy for gene editing in hematopoietic models.

This work was carried out in partnership with the BIGRes team (B-cell Ig Gene Remodeling Singularities), a joint EFS and Inserm laboratory led by Michel Cogné in Rennes, France, with significant contributions from Dr. Gregory Noel and support from Inside Therapeutics.

RESULTS
How do LNPs compare with electroporation for CRISPR delivery in HSCs?

Performance was evaluated in two stages. First, an eGFP mRNA model was used to assess dose-dependent RNA delivery in HSCs using a standard SM-102 LNP formulation. Second, a proof-of-concept study evaluated the delivery of Cas9 mRNA and sgRNA targeting the β2-microglobulin (B2M) locus. This knockout model was then used to compare LNP delivery directly with electroporation.

Additional details are provided in the Materials and Methods section below.

1: Physicochemical characterization of the LNP

Following formulation, the LNPs were characterized using NTA. Both the eGFP- and Cas9-containing LNPs showed mean particle sizes close to 100 nm, consistent with established benchmarks for efficient intracellular delivery (Figure 1).

2: Hematopoietic stem cells transfection

A. eGFP mRNA delivery via LNP in HSCs

HSCs were treated with eGFP-encapsulated RNA-LNPs formulated using the standard SM-102 formulation at increasing RNA doses of 0.125, 0.25, and 0.5 µg per 200,000 HSC cells.

Figure 2 shows the transfection results, measured by the percentage of cells that became eGFP-positive.

Both approaches delivered similarly high knockout efficiencies, reaching around 100% B2M disruption under their optimal conditions. The key difference was cell viability. As shown in Figure 7, electroporation caused substantial cell loss, with around 20% cell death, while LNP-mediated delivery preserved near-complete viability at approximately 99%.

This difference is especially important in applications where cell recovery is limited, such as primary HSC editing for transplantation.

LNPs also used CRISPR reagents far more efficiently. Because the two approaches used different Cas9 formats—mRNA for LNP delivery and protein for electroporation—direct Cas9 dose comparisons were not made. However, sgRNA use per million cells showed a clear advantage for LNP delivery. Electroporation required around 3.2 µg of sgRNA per million cells, while LNP-mediated delivery achieved comparable editing with only 0.32 µg per million cells, representing an order-of-magnitude reduction.

This lower RNA requirement can reduce reagent costs, improve scalability, and support future GMP manufacturing and translational applications.

DISCUSSION
Can LNPs match electroporation for CRISPR editing in HSCs?

This study shows that hematopoietic stem cells can be effectively transfected with both eGFP mRNA and CRISPR reagents using a standard SM-102 lipid nanoparticle formulation. LNPs produced with the TAMARA microfluidic system delivered strong physicochemical performance, high transfection efficiency, a clear dose-response profile, and near-zero cytotoxicity.

For CRISPR-Cas9 delivery, LNPs achieved knockout levels comparable to electroporation while maintaining almost complete cell viability. This is a significant advantage for workflows involving sensitive primary cells, especially where cell recovery and functional integrity are critical.

LNP-based delivery also used far less sgRNA per cell than electroporation, demonstrating a major improvement in reagent efficiency.

Overall, these results support LNP-mediated delivery as a gentler, more scalable, and more translationally relevant alternative to electroporation for gene-editing workflows in hematopoietic models. While electroporation is limited to ex vivo manipulation, LNPs are also compatible with in vivo administration. This creates new opportunities for direct HSC-targeted editing and broader therapeutic strategies that electroporation-based approaches cannot support.

There is still room to improve LNP-mediated CRISPR editing further. Next-generation ionizable lipids, such as LP-01 originally developed by Intellia, may improve endosomal escape and increase editing efficiency. Additional optimization of delivery strategy—whether through co-delivery of Cas9 mRNA and sgRNA or by adjusting the timing of separate delivery for each component—may also reveal kinetic advantages that improve performance.

Future studies combining advanced lipid chemistries, optimized LNP architectures, and targeted delivery strategies may further elevate CRISPR editing in HSCs, potentially exceeding current electroporation benchmarks while preserving superior viability and translational potential.

MATERIALS USED
Summary of materials and methods used in this study

RNA-LNP Formulation

RNA-LNP composition

eGFP RNA : eGFP mRNA cleancap
Cas9 mRNA: Codon-optimized Cas9 mRNA
sgRNA: Proprietary design by the BIGRes team Lipid Composition : Moderna like composition including SM-102 ionizable lipid.
The composition can be found on the Inside Therapeutics website in the LNP starter kits.

RNA-LNP formulation systems: TAMARA

TAMARA is an advanced RNA-LNP formulation system developed by Inside Therapeutics.

Built on microfluidic technology, the platform supports the full R&D workflow for RNA-LNP medicines and therapies, from early screening through to in vivo testing. Its reusable chip features an optimized fluidic design that removes dead volumes, while high-performance microfluidic mixing delivers strong encapsulation efficiency with excellent control and repeatability of the LNPs produced. As a result, TAMARA provides an efficient and cost-effective solution for RNA-LNP development.

TAMARA also offers two microfluidic mixing approaches within a single chip: an optimized Herringbone mixer and a Baffle mixer. In this study, the Herringbone mixing approach was used throughout.

Formulation protocol

A standard formulation protocol was used for the formulation of RNA-LNP. Formulation parameters used:

• FRR: 1:3
• TFR: 5 mL/min
• Post formulation process: Ultrafiltration using amicon filters

More details on the formulation protocol can be found in the TAMARA standard protocol available on Inside Therapeutics website.

Characterization

RNA-LNP size

RNA-LNP size measurement was carried out using an NTA (Nanoparticle tracking analysis).

Cell assays

T -1: Transfection Day -1: 200,000 HSCs were seeded in the appropriate well plate, such that the cells were at 70-80% confluent on the end point.

T0: Transfection Day:

• The culture media was removed from the cells
• mRNA-LNP was added at the specified RNA concentration
• The cells were then incubated with the treatment for 24 hours

Flow cytometry

After transfection, protein expression was assessed using FACS.