Scanning mutagenesis of the voltage-gated sodium channel Na V 1.2 using base editing.

Autor: Pablo JLB; 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., Cornett SL; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., Wang LA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., Jo S; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., Brünger T; Cologne Center for Genomics, University of Cologne, 51149 Cologne, Germany; Genomic Medicine Institute, Lerner Research Institute, Neurological Institute, Cleveland Clinic, Cleveland, OH 44195, USA., Budnik N; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., Hegde M; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., DeKeyser JM; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA., Thompson CH; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA., Doench JG; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA., Lal D; 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., George AL Jr; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA., Pan JQ; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: jpan@broadinstitute.org.
Jazyk: angličtina
Zdroj: Cell reports [Cell Rep] 2023 Jun 27; Vol. 42 (6), pp. 112563. Date of Electronic Publication: 2023 Jun 01.
DOI: 10.1016/j.celrep.2023.112563
Abstrakt: It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (Na V s) 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 V 1.2 to identify more than 100 gRNAs that change Na V 1.2 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 V 1.2 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 V 1.2.
Competing Interests: Declaration of interests A.L.G. consults for and receives sponsored research funding from Praxis Precision Medicines and Neurocrine Biosciences for unrelated work.
(Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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