Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery

Abstract

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD_7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

Figure 1

(A) Chemical structure of amphiphilic xenopeptides for Cas9 RNP delivery. “Stp number” indicates the number of ionizable Stp building units at each side of the lysine. (B) eGFP knockout efficiency and cell viability of HeLa eGFP/tub cells after 48 h treatment with Cas9 RNP nanocarriers at 75 nM RNP dose. Fatty acid residues are indicated by color code. (C) eGFP knockout efficiency of HeLa eGFP/tub cells after 48 h treatment with Cas9 RNP nanocarriers at a series of RNP concentrations ranging from 1 nM to 100 nM. Data are presented as mean ± SD (n = 3).

Figure 2

(A) General synthetic route of artificial amino acid building blocks with protective groups for use in solid-phase peptide synthesis (SPPS). (B) Amphiphilic xenopeptide architectures selected for the construction of hydrophobically balanced carriers.

Figure 3

Dose titration of amphiphilic xenopeptides and logD_7.4 correlation with eGFP knockout efficiencies. (A) eGFP knockout efficiency in HeLa eGFP/tub cells after 48 h treatment with Cas9 RNP nanocarriers at a series of RNP concentrations between 0.1 and 100 nM. Data are presented as mean ± SD (n = 3). (B) Plot of eGFP knockout EC50 values versus logD_7.4 values of each xenopeptide series.

Figure 4

(A) Effect of dilution on the nanocarrier size determined by DLS; value = 0 indicates “not detectable”. (B) Nanocarrier stability against different concentrations of heparin (0, 0.25, 0.5, 1, 2.5, and 5 IU/μg sgRNA). Ribogreen was used for the detection of free RNA. (C) Plot of median fluorescence intensities (MFI) of ATTO647N-Cas9 protein and ATTO488-sgRNA determined by flow cytometry versus xenopeptide logD_7.4 values of each xenopeptide series. (D) Endocytosis pathway study with different inhibitors. Sodium azide: energy-dependent endocytosis; chlorpromazine: clathrin-mediated endocytosis; sucrose: clathrin-mediated endocytosis; nystatin: caveolae-mediated endocytosis; amiloride: macropinocytosis. Data are presented as mean ± SD (n = 3). Statistical analysis was performed by comparing each treatment group with the corresponding “no inhibitor” group. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. (E) Confocal laser scanning microscopy (CLSM) images of HeLa WT cells 4 h after treatment with the selected Cas9 RNP nanocarriers (75 nM RNP) containing 20% of ATTO647N-Cas9 and 20% ATTO488-sgRNA. Nuclei were stained with DAPI (blue). The merged channel indicates co-localization (yellow) of ATTO647N-Cas9 (red) and ATTO488-sgRNA (green). (F) CLSM images of HeLa mRuby3/gal8 cells treated with the selected Cas9 RNP nanocarriers (75 and 5 nM RNP) for 4 h. Nuclei were stained with DAPI (blue). Red punctuate mRuby3/gal8 spots indicate endosomal membrane damage.

Figure 5

(A) Schematic illustration of eGFP to BFP conversion by co-delivery of Cas9 RNP and an ssDNA template into eGFP expressing cells. (B) Heat maps of NHEJ, HDR, total edited, and nonedited percentages in HeLa GFPd2 cells 48 h after treatment with Cas9 RNP/ssDNA nanocarriers (TFE-IDAtp1-LinA) at varied RNP concentrations and RNP/ssDNA ratios. (C) Heat map of HDR percentages in HeLa GFPd2 cells 48 h after treatment with different lipopeptide-based Cas9 RNP/ssDNA (fixed at 1/4) nanocarriers at varied RNP concentrations.