使用策划的基因座引物编辑进行多路变异效应测定的协议。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-05-25 DOI:10.1016/j.xpro.2025.103851

Carina G Biar, Nicholas Bodkin, Gemma L Carvill, Jeffrey D Calhoun

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引用次数: 0

摘要

变异效应的多重分析（MAVEs）可以同时对许多变异进行表征。在这里，我们提出了一种使用策划位点引物编辑（cliPE）执行MAVEs的方案，这是一种可访问的实验管道，可以对目标基因进行引物编辑。我们描述了设计主要编辑试剂，筛选基因组编辑效率，选择编辑的细胞池以容纳不同的遗传变异和测序的步骤。最后，我们详细介绍了进行富集分析以识别具有正常或异常活性的变体的程序。

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Protocol to perform multiplexed assays of variant effect using curated loci prime editing.

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.

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