Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing
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 Na...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-06-01
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| Series: | Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124723005740 |
| _version_ | 1797812817232920576 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Juan Lorenzo B. Pablo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T07:44:01Z |
| format | Article |
| id | doaj.art-a26ee4757b7c41068a33d54562302016 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-03-13T07:44:01Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-a26ee4757b7c41068a33d545623020162023-06-03T04:21:55ZengElsevierCell Reports2211-12472023-06-01426112563Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editingJuan Lorenzo B. Pablo0Savannah L. Cornett1Lei A. Wang2Sooyeon Jo3Tobias Brünger4Nikita Budnik5Mudra Hegde6Jean-Marc DeKeyser7Christopher H. Thompson8John G. Doench9Dennis Lal10Alfred L. George, Jr.11Jen Q. Pan12Stanley 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 authorStanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USAStanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USAStanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USACologne Center for Genomics, University of Cologne, 51149 Cologne, Germany; Genomic Medicine Institute, Lerner Research Institute, Neurological Institute, Cleveland Clinic, Cleveland, OH 44195, USAStanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USABroad Institute of MIT and Harvard, Cambridge, MA 02142, USADepartment of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USADepartment of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USABroad Institute of MIT and Harvard, Cambridge, MA 02142, USACologne 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, USADepartment of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USAStanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2211124723005740CP: Neuroscience |
| spellingShingle | 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 Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing Cell Reports CP: Neuroscience |
| title | Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing |
| title_full | Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing |
| title_fullStr | Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing |
| title_full_unstemmed | Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing |
| title_short | Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing |
| title_sort | scanning mutagenesis of the voltage gated sodium channel nav1 2 using base editing |
| topic | CP: Neuroscience |
| url | http://www.sciencedirect.com/science/article/pii/S2211124723005740 |
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