Abstract
CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in place, the system allows investigators to produce their own neuronal cell models for the inducible expression of any coding sequence in less than a month. Due to the availability of preinserted enhanced green fluorescent protein (EGFP) coding sequences that can be fused to the protein-of-interest, the system facilitates functional investigations that track a protein-of-interest by live-cell microscopy as well as interactome analyses that capitalize on the availability of exquisitely efficient EGFP capture matrices.