Centre Hospitalier Universitaire de Québec Research Center-Université Laval, Québec, QC, G1V 4G2, Canada.
Université Laval Cancer Research Centre, Québec, QC, G1V 0A6, Canada.
Nat Commun. 2022 Oct 7;13(1):5909. doi: 10.1038/s41467-022-33669-z.
Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na/K ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.
Prime 编辑技术可在无需供体 DNA 模板的情况下实现精确的点突变、小片段插入或短片段缺失。然而，在广泛的人类细胞类型中，效率仍然是一个关键挑战。在这项工作中，我们通过共编辑普遍存在且必需的钠/钾泵（Na/K ATPase）来设计一种稳健的共选择策略。我们可以轻松地对高度修饰的细胞池和克隆进行共编辑，以进行功能研究，同时最小化 pegRNA 的优化。这一过程表明，对未编辑链进行切口会刺激多等位基因编辑，但通常会在靶位点产生串联重复和大片段缺失，这种结果是由原间隔相邻基序的相对取向决定的。我们的方法简化了具有多种遗传修饰的细胞系的生产，为生物学研究创建了细胞模型，并为开发特定于细胞类型的共选择策略奠定了基础。
