Department of Chemistry and Biotechnology, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, Hawthorn, VIC, Australia.
Department of Neuroscience, Central Clinical School, Monash University, Melbourne, VIC, Australia.
Methods Mol Biol. 2022;2495:99-114. doi: 10.1007/978-1-0716-2301-5_6.
CRISPR/Cas9 system is a powerful genome-editing technology for studying genetics and cell biology. Safe harbor sites are ideal genomic locations for transgene integration with minimal interference in cellular functions. Gene targeting of the AAVS1 locus enables stable transgene expression without phenotypic effects in host cells. Here, we describe the strategy for targeting the AAVS1 site with an inducible Neurogenin-2 (Ngn2) donor template by CRISPR/Cas9 in hiPSCs, which facilitates generation of an inducible cell line that can rapidly and homogenously differentiate into excitatory neurons.
CRISPR/Cas9 系统是一种强大的基因组编辑技术，可用于研究遗传学和细胞生物学。安全港位点是转基因整合的理想基因组位置，对细胞功能的干扰最小。AAVS1 基因座的基因靶向使转基因在宿主细胞中稳定表达，而不会产生表型效应。在这里，我们描述了通过 CRISPR/Cas9 在 hiPSCs 中靶向 AAVS1 位点的诱导性神经基因 2 (Ngn2) 供体模板的策略，该策略有助于生成一种诱导性细胞系，该细胞系可以快速且均匀地分化为兴奋性神经元。
