Osgood Nicola R B, Zawalick Natalie M, Sawyer Courtney B, Cowan Quinn T, Gu Sifeng, Mawson S J, Ranzau Brodie L, Li Lehan, Gymrek Melissa, Goren Alon, Komor Alexis C
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, United States.
Department of Medicine, University of California San Diego, La Jolla, CA, United States.
Methods Enzymol. 2025;712:351-404. doi: 10.1016/bs.mie.2025.01.001. Epub 2025 Feb 5.
Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.
在过去十年中，基因组编辑已引起广泛关注，导致基因组工程工具箱大幅扩展。碱基编辑器是一类工具，可在不使用双链断裂的情况下在基因组DNA中安装单核苷酸变化。随着新的和/或改进的碱基编辑器系统不断发展，丰富的选择很容易让人应接不暇。在此，我们提供明确的指导，以促进碱基编辑器的选择并设计适合各种需求的引导RNA（gRNA）。此外，我们详细描述了如何生成gRNA质粒、转染各种哺乳动物细胞类型以及评估编辑效率。最后，我们针对碱基编辑过程中遇到的一些常见问题给出替代方法和故障排除提示。
