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

doi:10.1016/j.celrep.2023.112563

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

Cell Rep. 2023 Jun 27;42(6):112563. doi: 10.1016/j.celrep.2023.112563. Epub 2023 Jun 1.

Authors

Affiliations

¹ 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. Electronic address: jpablo@broadinstitute.org.
² Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
³ Cologne Center for Genomics, University of Cologne, 51149 Cologne, Germany; Genomic Medicine Institute, Lerner Research Institute, Neurological Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁵ Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
⁶ 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.
⁷ Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: jpan@broadinstitute.org.

Abstract

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.

Keywords: CP: Neuroscience; CRISPR; NaV1.2; base editing; channelopathy; electrophysiology; ion channels; mutational screening; neurodevelopmental disorder; variants to function; voltage-gated sodium channel.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Gene Editing* / methods
Mutagenesis / genetics
Mutation
Mutation, Missense / genetics
NAV1.2 Voltage-Gated Sodium Channel*
Voltage-Gated Sodium Channels*

Substances

Voltage-Gated Sodium Channels
NAV1.2 Voltage-Gated Sodium Channel

Abstract

Publication types

MeSH terms

Substances

Grants and funding