Scanning Mutagenesis of the Voltage-Gated Sodium Channel NaV1.2 Using Base Editing

Authors

Juan Lorenzo B Pablo
Savannah L Cornett
Lei A Wang
Sooyeon Jo
Tobias Brünger
Nikita Budnik
Mudra Hegde
Jean-Marc DeKeyser
Christopher H Thompson
John G Doench
Dennis Lal
Alfred L George
Jen Q Pan

Publication Date

6-27-2023

Journal

Cell Reports

Abstract

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 screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 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 NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Keywords

Gene Editing, Mutagenesis, Mutation, Mutation, Missense, Voltage-Gated Sodium Channels, NAV1.2 Voltage-Gated Sodium Channel

DOI

10.1016/j.celrep.2023.112563

PMID

37267104

PMCID

PMC10592450

PubMedCentral® Posted Date

10-23-2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes