Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Int J Mol Sci. 2022 Oct 7;23(19):11919. doi: 10.3390/ijms231911919.
CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.
CRISPR 技术为编辑细胞基因组提供了一种简单而强大的方法，为基础研究和医学提供了强大的工具。虽然使用 Cas9 靶向基因组位点非常有效，但在该位点进行特定突变的效率要低得多，因为这取决于内源性 DNA 修复机制。已经开发了各种策略来提高基因敲入诱变的效率，但通常所需的细胞仍然只占总群体的一小部分。为了提高效率，已经开发了选择编辑细胞的策略。在某些应用中，可选择的外源基因直接与感兴趣的基因（GOI）相连。或者，共编辑，其中 GOI 与可选择的基因一起编辑，富集所需的细胞，因为成功编辑可选择基因的细胞很可能也编辑了 GOI。为了最小化宿主基因组的干扰，已经开发了“无疤痕”选择策略，其中修饰的细胞仅在 GOI 中发生突变。在这篇综述中，我们将讨论用于提高哺乳动物细胞特定基因组编辑的策略，重点讨论如何选择成功编辑的细胞。
