genome editing in single mammalian brain neurons through CRISPR-Cas9 and cytosine base editors.

Gene manipulation is a useful approach for understanding functions of genes and is important for investigating basic mechanisms of brain function on the level of single neurons and circuits. Despite the development and the wide range of applications of CRISPR-Cas9 and base editors (BEs), their implementation for an analysis of individual neurons remained limited. In fact, conventional gene manipulations are generally achieved only on the population level. Here, we combined either CRISPR-Cas9 or BEs with the targeted single-cell electroporation technique as a proof-of-concept test for gene manipulation in single neurons . Our assay consisted of CRISPR-Cas9- or BEs-induced gene knockout in single Purkinje cells in the cerebellum. Our results demonstrate the feasibility of both gene editing and base editing in single cells in the intact brain, providing a tool through which molecular perturbations of individual neurons can be used for analysis of circuits and, ultimately, behaviors.