Kiesler Patricia, Lee Stella S, Norris Alexis L, Miller Mayumi F, Mercado Carlo J, Moyer Adam L, Maragh Samantha
Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Center for Veterinary Medicine, Office of New Animal Drug Evaluation, US Food and Drug Administration, Rockville, MD 20855, USA.
STAR Protoc. 2024 Dec 20;5(4):103385. doi: 10.1016/j.xpro.2024.103385. Epub 2024 Oct 10.
Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.
基因组编辑技术正被用于动物身上以实现多种目的，包括改善动物健康和公共卫生成果。对基因组编辑试剂和预期的基因组改变进行表征是培育编辑动物的关键一步。在此，我们展示了一种在猪睾丸（ST）细胞系中进行基因组编辑的方案。我们描述了在体外评估CRISPR-Cas9复合物功能、通过转染将编辑分子导入细胞以及通过桑格测序评估靶点编辑的步骤。
