Fig. 1: A modular approach was developed to enable systemic nanoparticle delivery of CRISPR-Cas9 RNPs for tissue-specific genome editing.

a Addition of a permanently cationic supplemental component (e.g., DOTAP) into traditional LNP formulations enabled encapsulation and protection of Cas9/sgRNA complexes using neutral buffers during nanoparticle formation. Precise tuning of the DOTAP percentage mediated tissue-specific gene editing. b Size distribution of Cas9/sgLuc RNPs prepared in PBS buffer (pH 7.4) and citrate buffer (pH 4.0). The size increase is likely due to denaturization. c Size distribution of 5A2-DOT-10 encapsulating Cas9/sgLuc RNPs prepared in PBS and citrate buffer. 5A2-DOT-10 prepared without RNPs was used as control. d Size distribution of Cas9/sgRNA RNPs with Cas9/sgLuc molar ratio of 1/1, 1/3, and 1/5. e Size distribution of 5A2-DOT-10 encapsulating Cas9/sgLuc with molar ratio of 1/1, 1/3, and 1/5. f Zeta potential of Cas9/sgRNA RNPs showing decreasing charge. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). g No significant difference of zeta potential was observed for 5A2-DOT-10 encapsulating Cas9/sgLuc with different molar ratios. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). h Time-dependent cellular uptake of 5A2-DOT-10 LNPs encapsulating EGFP-fused Cas9/sgRNAs showing cytoplasmic release and gradual entry into the nucleus (n = 3 biologically independent samples). Scale bar: 10 μm. Red arrows show distribution of EGFP-fused Cas9/sgRNAs inside cells. i Inhibition of 5A2-DOT-10 LNP uptake was studied using specific endocytosis inhibitors. Amiloride (AMI): inhibitor of macropinocytosis; chlorpromazine (CMZ): inhibitor of clathrin-mediated endocytosis; Genistein (GEN): inhibitor of caveolae-mediated endocytosis; Methyl-β-cyclodextrin (MβCD): lipid rafts-mediated endocytosis; 4 degree: energy-mediated endocytosis. Data are presented as mean ± s.e.m. (n = 3 biologically independent samples). Source data are in the Source Data file.