Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
KG. Jebsen Centre for B cell Malignancies, University of Oslo, Oslo, Norway.
Methods Mol Biol. 2020;2115:445-454. doi: 10.1007/978-1-0716-0290-4_25.
Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.
真核生物的基因组编辑技术通过靶向编辑工具的应用得到了极大的改善。CRISPR/Cas9 技术的发展促进了哺乳动物细胞的基因组编辑。然而，将 CRISPR 组件有效地递送到悬浮生长的细胞中仍然是一个挑战。在这里，我们提出了一种将两个必需成分 Cas9 和 sgRNA 顺序递送到 B 淋巴细胞系中的策略。通过逆转录病毒转导获得稳定的 Cas9 表达，然后将 sgRNA 瞬时递送到 Cas9+细胞中。这种方法提高了基因组编辑的靶向效率，并通过 sgRNA 的短暂存在降低了潜在的脱靶位点。目前的方法可以很容易地应用于其他用 CRISPR/Cas9 难以编辑的细胞类型。
