Abstract

Efficient Somatic in vivo Gene Editing using CRISPR/Cas9

Thierry VandenDriessche, Group Leader, Free University of Brussel (VUB)

The robustness of liver-directed gene editing could be substantially improved by enhancing Cas9 expression in the liver. To achieve this, we developed and validated a new computational approach of rational in silico vector design. This approach relies on a genome-wide bio-informatics strategy to identify cis-acting regulatory modules (CRMs) containing evolutionary conserved clusters of transcription factor binding site motifs that determine high liver-specific gene expression. Incorporation of these CRMs into adeno-associated viral (AAV) and non-viral plasmid and transposon vectors enhanced gene expression in mice liver 10 to 100-fold, depending on the promoter used. We built further upon this concept of computational vector design to develop a robust platform for tissue-specific gene inactivation in the liver using CRISPR/Cas9. As a proof of concept, we demonstrated that liver-specific over-expression of Cas9 in combination with FIX-specific guide RNAs resulted in the selective inactivation of the endogenous murine FIX gene consistent with the emergence of a hemophilic phenotype. This opens new perspectives for the use of CRISPR/Cas9 for in vivo somatic genome engineering in specific target organs.