Designer DNA-binding proteins based on transcriptional activator-like effectors (TALEs) and zinc finger proteins (ZFPs) are easily tailored to recognize specific DNA sequences in a modular manner. They can be engineered to generate tools for targeted genome perturbation. Here, we review recent advances in these versatile technologies with a focus on designer nucleases for highly precise, efficient, and scarless gene modification. By generating double stranded breaks and stimulating cellular DNA repair pathways, TALE and ZF nucleases have the ability to modify the endogenous genome. We also discuss current applications of designer DNA-binding proteins in synthetic biology and disease modeling, novel effector domains for genetic and epigenetic regulation, and finally perspectives on using customizable DNA-binding proteins for interrogating neural function.