Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Abstract

Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing mRNA and other nucleic acids, especially large mRNA. Cas9 protein is the nuclease part of the powerful gene-editing tool, CRISPR/Cas9 system, Cas9 mRNA is particularly large, thus presents a big challenge for delivery. We assembled a multilayered biodegradable nanocarrier to load Cas9 mRNA inside to protect Cas9 mRNA from degradation. We used a microfluidic chip to synthesize a small, positively charged, and degradable core to attract negatively charged Cas9 mRNA. The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome. The multilayered nanocarriers elevated the delivery efficiency of Cas9 mRNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers. In addition, the multilayered nanocarriers do not require reduced serum medium for transfection. When using the standard complete medium for transfection, the multilayered nanocarriers could increase the expression of Cas9 mRNA by over 15 folds compared to commercially used non-viral carriers. The co-delivery of Cas9 mRNA and sgRNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers. Based on the higher transfection efficiency of Cas9 mRNA/sgRNA than commercially used non-viral carriers, these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 mRNA/sgRNA and other nucleic acids.