Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

• Main Authors: Addlestone, E. (Author), Ates, I. (Author), Cottle, R.N (Author), Fernando, L. (Author), Lee, C.M (Author), Rathbone, T. (Author), Richards, V.P (Author)
• Format: Article
• Language: English
• Published: Mary Ann Liebert Inc. 2022
• Online Access: View Fulltext in Publisher

 Adeno-Associated virus vectors are the most used delivery method for liver-directed gene editing. Still, they are associated with significant disadvantages that can compromise the safety and efficacy of therapies. Here, we investigate the effects of electroporating CRISPR-Cas9 as mRNA and ribonucleoproteins (RNPs) into primary hepatocytes regarding on-Target activity, specificity, and cell viability. We observed a transfection efficiency of >60% and on-Target insertions/deletions (indels) of up to 95% in primary mouse hepatocytes electroporated with Cas9 RNPs targeting Hpd, the gene encoding hydroxyphenylpyruvate dioxygenase. In primary human hepatocytes, we observed on-Target indels of 52.4% with Cas9 RNPs and >65% viability after electroporation. These results establish the impact of using electroporation to deliver Cas9 RNPs into primary hepatocytes as a highly efficient and potentially safe approach for therapeutic liver-directed gene editing and the production of liver disease models. © Tanner Rathbone et al. 2022; Published by Mary Ann Liebert, Inc. 2022.