Scanning mutagenesis of the voltage-gated sodium channel Na_V1.2 using base editing

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (Na_Vs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling Na_V1.2 to identify more than 100 gRNAs that change Na_V1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in Na_V1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in Na_V1.2.