| Autor: |
Rathbone T; Department of Bioengineering, University College Cork, Cork, Ireland., Ates I; Department of Bioengineering, University College Cork, Cork, Ireland., Fernando L; Department of Bioengineering, University College Cork, Cork, Ireland., Addlestone E; Department of Bioengineering, University College Cork, Cork, Ireland., Lee CM; APC Microbiome Ireland, University College Cork, Cork, Ireland., Richards VP; Department of Biological Sciences, Clemson University, Clemson, South Carolina., Cottle RN; Department of Bioengineering, University College Cork, Cork, Ireland. |
| Abstrakt: |
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. |
