It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screened a library of 368 guide RNAs (gRNA) tiling NaV1.2 to identify more than 100 gRNAs that changed NaV1.2 function. We sequenced base edits made by a subset of these gRNAs to confirm specific variants that drove changes in channel function. Electrophysiological characterization of these channel variants validated the screen results and provided functional mechanisms of channel perturbation. The majority of the changes caused by these gRNAs were classified as loss-of-function along with two missense mutations that led to gain-of-function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies the largest set of loss-of-function mutations in a single NaV1.2 study to date.

The folder "demultiplexed_sequencing_data" contains all the demultiplexed sequencing reads in FASTQ format. Each fastq file contains sequencing reads from a sample. The experimental condition associated with each sample, as well as the amplicon sequence and guide sequence, are listed in "batch_file.txt."