使用经过整理的位点进行碱基编辑来进行变异效应多重分析的方案。
Protocol to perform multiplexed assays of variant effect using curated loci prime editing.
作者信息
Biar Carina G, Bodkin Nicholas, Carvill Gemma L, Calhoun Jeffrey D
机构信息
Ken and Ruth Davee Department of Neurology, Northwestern Feinberg School of Medicine, Chicago, IL 60611, USA; Genome Sciences, University of Washington, Seattle, WA 98195, USA.
Ken and Ruth Davee Department of Neurology, Northwestern Feinberg School of Medicine, Chicago, IL 60611, USA.
出版信息
STAR Protoc. 2025 Jun 20;6(2):103851. doi: 10.1016/j.xpro.2025.103851. Epub 2025 May 25.
Multiplexed assays of variant effect (MAVEs) perform simultaneous characterization of many variants. Here, we present a protocol to perform MAVEs using curated loci prime editing (cliPE), an accessible experimental pipeline that enables prime editing of a target gene. We describe steps for designing prime editing reagents, screening for genome editing efficiency, selecting a pool of cells edited to harbor different genetic variants, and sequencing. Lastly, we detail procedures for performing enrichment analysis to identify variants with normal or aberrant activity.
变异效应多重分析（MAVEs）可对多个变异进行同时表征。在此，我们展示了一种使用经过精心设计的位点引导编辑（cliPE）进行MAVEs的方案，这是一种可实现目标基因引导编辑的便捷实验流程。我们描述了设计引导编辑试剂、筛选基因组编辑效率、选择编辑后携带不同遗传变异的细胞池以及测序的步骤。最后，我们详细介绍了进行富集分析以鉴定具有正常或异常活性变异的程序。
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