Abstract
The CRISPR/cas9 system facilitates sited-directed mutagenesis in DNA by using a single gRNA to recruit cas9 endonuclease to the target site. Even though the CRISPR/cas9 system has been widely applied in a variety of cancer and embryonic cell lines, its applications in post-mitotic cells are still limited. Here we first evaluated targeting efficacy of six gRNA targeting the mCitrine gene in cell cultures. 1/3 of gRNAs showed high on-target activity and turned off mCitrine expression. We then delivered lentiviral vectors carrying genes encoding cas9 and gRNAs into cortical slice culture. The delivered CRISPR/cas9 system successfully disrupted the endogenous tdTomato gene expression in post-mitotic cells. Our result suggest that optimized gRNAs requires both low off-target effects and high on-target activity. We also confirmed that the CRISPR/cas9 system is effective in post-mitotic cells and application is possible in cortical slice culture.