PDF(Pubmed)
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.
摘要：
将传统的突变扫描应用于电压门控钠通道（Navs）并在功能上注释这些基因中的大量编码变体是具有挑战性的。使用胞嘧啶碱基编辑器和合并的活力测定，我们筛选了368个指导RNA（gRNA）的文库，以鉴定超过100个改变NaV1.2功能的gRNA。我们对由这些gRNA的子集进行的碱基编辑进行测序，以确认驱动通道功能变化的特定变体。这些通道变体的电生理表征验证了筛选结果并提供了通道扰动的功能机制。由这些gRNA引起的大多数变化可归类为功能丧失以及导致NaV1.2通道功能获得的两个错义突变。这种在功能上大规模表征离子通道蛋白变体的两级策略识别了NaV1.2中的大量功能丧失突变。
