For in vitro genome editing experiments, transient transfection was performed using the jetOPTIMUS following Polyplus’s instructions. For the sFLEx system, Neuro-2a cells (N2A cells) were transfected with a recombinase expressing plasmids (pCAG-Cre) and a recombinase-inducible, SpCas9-expressing plasmid (pAAV-pCALM1-sFLEx-HA-SpCas9- polyA-miniU6-sgRNAShank3). In cases where pAAV-pCALM1-SpCas9-polyA-miniU6-sgRNAShank3 was tested, only one plasmid was transfected. Cells were harvested 72 h after transfection and editing was confirmed by performing Sanger sequencing on individual clones prepared using Topo cloning of the PCR product of interest.

Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120 bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional in vivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.