RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Nat Methods. 2023 Jun;20(6):898-907. doi: 10.1038/s41592-023-01859-2. Epub 2023 May 8.
Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.
主导编辑具有广泛的潜在研究和临床应用。然而，用于描绘其全基因组编辑活动的方法通常依赖于间接的全基因组编辑评估或近同源序列的计算预测。在这里，我们描述了一种用于鉴定潜在主导编辑器脱靶位点的全基因组方法，我们称之为 PE-tag。该方法依赖于在主导编辑器活性位点附加或插入扩增标签，以允许识别它们。PE-tag 可使用提取的基因组 DNA、哺乳动物细胞系和成年小鼠肝脏在体外对脱靶位点进行全基因组分析。PE-tag 组件可以以多种格式用于脱靶位点检测。我们的研究与先前描述的主导编辑器系统的高特异性一致，但我们发现脱靶编辑率受主导编辑向导 RNA 设计的影响。PE-tag 代表了一种易于使用、快速和敏感的方法，可用于全基因组鉴定主导编辑器的活性和评估主导编辑器的安全性。
