Sanjurjo-Soriano Carla, Erkilic Nejla, Mamaeva Daria, Kalatzis Vasiliki
Institute for Neurosciences of Montpellier, Inserm, Montpellier, France.
University of Montpellier, Montpellier, France.
Methods Mol Biol. 2022;2454:589-606. doi: 10.1007/7651_2021_362.
The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.
2006年开发出了将体细胞重编程为诱导多能干细胞（iPSC）的技术，这代表着干细胞研究的一项重大突破。2013年，生物医学研究取得了又一个里程碑式的进展，当时CRISPR/Cas9系统被用于编辑哺乳动物细胞的基因组。人（h）iPSC与CRISPR/Cas9技术的结合为细胞治疗和再生医学带来了巨大希望。然而，包括时间和劳动力消耗、系统的效率和功效以及Cas9核酸酶诱导的潜在脱靶效应等几个限制因素仍有待解决。在此，我们描述了一种利用核转染介导的方案将CRISPR/Cas9组分导入细胞，从而轻松地对hiPSC进行基因改造的详细方法，并讨论了设计实验时需要考虑的关键点。通过我们的方法产生的克隆的、经过基因组编辑的hiPSC系可直接用于下游应用。
