Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing
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, Jr.,
Jen Q. Pan
Affiliations
Juan Lorenzo B. Pablo
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Corresponding author
Savannah L. Cornett
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Lei A. Wang
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Sooyeon Jo
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Tobias Brünger
Cologne Center for Genomics, University of Cologne, 51149 Cologne, Germany; Genomic Medicine Institute, Lerner Research Institute, Neurological Institute, Cleveland Clinic, Cleveland, OH 44195, USA
Nikita Budnik
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Mudra Hegde
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Jean-Marc DeKeyser
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
Christopher H. Thompson
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
John G. Doench
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
Dennis Lal
Cologne Center for Genomics, University of Cologne, 51149 Cologne, Germany; Genomic Medicine Institute, Lerner Research Institute, Neurological Institute, Cleveland Clinic, Cleveland, OH 44195, USA; Department of Neurology, McGovern Medical School, UTHealth, Houston, TX 77030, USA
Alfred L. George, Jr.
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
Jen Q. Pan
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Corresponding author
Summary: 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.
