Better Cell Entry of Small Cas Proteins: Comparing Cas12f and Cas9 in Human Cells

Human embryonic kidney (HEK293T) cells were obtained from ATCC and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma–Aldrich, USA) and 1% penicillin‐streptomycin (Thermo Fisher Scientific, USA). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Recombinant Cas9 protein was purchased from New England Biolabs (NEB), USA. pET28a‐SUMO‐AsCas12f‐2NLS was a gift from Quanjiang Ji (Addgene plasmid # 171613) [10]. Cas12f was expressed in BL21(DE3) Escherichia coli and purified by the Ni‐nitrilotriacetic acid (NTA)‐based chromatography (Figure S1). Guide RNAs targeting a gene sequence were synthesized by Integrated DNA Technologies (IDT), USA, and are indicated in Table S1. The RNP complexes were prepared by incubating Cas9 or Cas12f proteins with sgRNA at a molar ratio of 1:1.1 in nuclease‐free duplex buffer (IDT, USA) at 25°C (Cas9) or 45°C (Cas12f) for 10 min.

PepFect14 (PF14) peptide was purchased from Pepscan, USA. Cas9/PF14 and Cas12f/PF14 complexes were formed by adding PF14 at the desired molar ratios to 6 pmol RNPs in a total of 100 µL HEPES‐buffered glucose solution (20 mM HEPES, 5% glucose, pH = 7.2) and vortexing immediately for 2 s. The mixture was incubated for 40 min at room temperature to form RNP/PF14 complexes.

Complexes prepared for transfection were applied directly to a TEM grid. Complexes containing 2.5 µg of Cas protein were deposited onto a carbon‐coated TEM grid (300 mesh) and incubated for 2 min. The grids were then fixed with 0.5% glutaraldehyde, rinsed with nuclease‐free distilled deionized water, and stained with 2% uranyl acetate for 1 min. After air drying, the grids were analyzed.

HEK293T cells were seeded at a density of 15,000 cells per well in 100 µL FreeStyle293 medium (Thermo Fischer Scientific, USA) in a 96‐well plate 24 h before transfection. For peptide transfection, 20 µL of RNP/PF14 complexes containing 1.2 pmol RNP (RNP:PF14 molar ratio 1:100) were added to the cells. For transfection using lipofectamine, Lipofectamine CRISPRMAX (Thermo Fisher Scientific, USA) was used to transfect 1.2 pmol RNP, according to manufacturer's instructions. After 4 h, cells were topped up with 100 µL FreeStyle293 media containing 20% FBS.

RNP/PF14 complexes (RNP:PF14 molar ratio 1:100) were formed as described above and added to cells at the indicated amounts. MTT reagent (Sigma–Aldrich, USA) was added 24 h after transfection and assay was carried out according to manufacturer's instructions. Absorbance was then measured at 570 nm using a plate reader.

RNP complexes were prepared as mentioned above with a gRNA fluorescently labeled with ATTO⁵⁵⁰ (IDT, USA) before transfection. After 6 and 24 h, cells were analyzed using a fluorescence microscope, and uptake of the ATTO⁵⁵⁰ fluorescently‐labeled gRNA was quantified using a BD FACSAria flow cytometer using a PE‐CF594 filter. Results were expressed as percentages of uptake and mean fluorescence intensity ratios (MFIR) (ratio of treated cells/untreated control).

Efficient delivery of Cas RNPs relies on vectors that ensure cellular internalization while preserving the functionality of the complexes. Non‐viral vectors, such as amphipathic peptides, have demonstrated potential as delivery agents due to their ability to form nanosized complexes and mediate cellular uptake through hydrophobic interactions. Among these, PepFect14 (PF14), an amphipathic peptide, has shown success in delivering Cas9 RNPs, achieving comparable efficiency to lipid‐based vectors like Lipofectamine [7, 8]. Through hydrophobic interactions, amphipathic peptides are not only capable of binding RNPs but also achieving levels of cellular internalization resembling lipid‐based transfection reagents. We used PF14 and Lipofectamine as efficient and different methods for delivery to compare the uptake of a smaller Cas protein, Cas12f, with the widely used larger Cas9 (Figure 1).

To test whether PF14 (net charge +5) could similarly bind and complex Cas12f RNPs and determine the optimal ratio for full complexing, PF14 was added to Cas12f RNPs at increasing molar ratios in a gel‐shift assay (Figure 2A). PF14 was able to bind and fully complex Cas9 RNPs at a molar ratio of 40, and Cas12f RNPs at a molar ratio of 80 as shown by the retention of RNPs to the wells of the gel (Figure 2A). The higher ratio required to fully neutralize the charge of Cas12f RNPs could be explained by the higher net negative charge (–202) carried by those RNPs compared with Cas9 RNPs (net charge –80).

To characterize and compare the biophysical properties of the Cas9/PF14 and Cas12f/PF14 complexes, particle size and zeta‐potential analyses were carried out (Figure 2B). Dynamic light scattering (DLS) showed that Cas12f RNPs, with a hydrodynamic diameter of ∼250 nm, were smaller in size compared to Cas9 RNPs (∼1100 nm). This is expected due to the smaller size of the Cas12f protein (∼51 kDa) compared to Cas9 (>150 kDa). At molar ratios 1:50 and above, Cas9/PF14 and Cas12f/PF14 complexes were condensed to a size of 150–200 nm and carried a positive charge (Figure 2B). Transmission electron microscopy (TEM) displayed the formation of Cas9/PF14 and Cas12f/PF14 particles of 50–120 nm in diameter at an RNP:PF14 molar ratio of 1:100 (Figures 2C and S2).

Cellular uptake events can start immediately after transfection [11, 12]. Thus, comparing the uptake efficiency of Cas9 and Cas12f is best done at the initial binding with cellular receptors, with the resolution decreasing as the receptors become saturated. We used flow cytometry to measure and compare the cellular uptake of equimolar amounts of Cas9 and Cas12f RNPs at 6 and 24 h post‐transfection in HEK293T cells. The Cas9 and Cas12f RNP/PF14 used were non‐toxic to cells as was shown by MTT assay (Figure S3). At 6 h, the percentage of cells that uptook Cas9 or Cas12f RNPs did not vary and represented ∼100% of cells with PF14 peptide transfection, and ∼45% with Lipofectamine transfection (Figure 3A). At 24 h, the percentages of cells with RNP uptake remained above 90% with PF14, and decreased only for Cas9% to 33% but remained at similar levels for Cas12f with Lipofectamine, indicating sustained presence of Cas12f in cells (Figure 3B).

At both 6 and 24 h, mean fluorescence intensity ratio (MFIR) analysis revealed significantly higher levels of RNP uptake in cells transfected with Cas12f compared to Cas9 using either PF14 or Lipofectamine, suggesting enhanced molar uptake of the smaller Cas12f protein (Figure 3Aii,Bii). At 24 h, MFIRs were reduced by ∼50%, indicating protein clearance, with Cas12f remaining at significantly higher levels than Cas9. This observation highlights the potential advantages of smaller sized cargoes in enhancing cellular uptake and improving the overall efficiency of CRISPR delivery systems.