Genome Engineering Human ESCs or iPSCs with Cytosine and Adenine Base Editors.
Genome Engineering Human ESCs or iPSCs with Cytosine and Adenine Base Editors.
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使用胞嘧啶和腺嘌呤碱基编辑器对人类 ESC 或 iPSC 进行基因组工程。
DOI：
10.1007/7651_2022_461
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Gadue,Paul
中科院分区：
文献类型：
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作者：
Pavani,Giulia;Klein,JoshuaG;French,DeborahL;Gadue,Paul
在人类胚胎干细胞（ECS）或诱导多能干细胞（iPSC）中设计特定突变的能力对于人类疾病建模和生物过程研究极其重要。虽然 CRISPR/Cas9 可以在 iPSC 的编码序列中稳健地产生基因敲除 (KO) 和基因位点修饰，但由于技术限制，产生单等位基因突变或修饰非编码序列中的特定核苷酸仍然很困难。在这里，我们描述如何利用胞嘧啶 (BE4max) 和腺嘌呤 (ABEmax) 碱基编辑器在 iPSC 中引入精确突变而不诱导 DNA双链断裂。本章阐述了如何利用碱基编辑技术设计和克隆 gRNA、评估编辑效率以及检测 iPSC 中特定位点的基因组编辑。
The ability to engineer specific mutations in human embryonic stem cells (ECSs) or induced pluripotent stem cells (iPSCs) is extremely important in the modeling of human diseases and the study of biological processes. While CRISPR/Cas9 can robustly generate gene knockouts (KOs) and gene loci modifications in coding sequences of iPSCs, it remains difficult to produce monoallelic mutations or modify specific nucleotides in noncoding sequences due to technical constraints.Here, we describe how to leverage cytosine (BE4max) and adenine (ABEmax) base editors to introduce precise mutations in iPSCs without inducing DNA double-stranded breaks. This chapter illustrates how to design and clone gRNAs, evaluate editing efficiency, and detect genomic edits at specific sites in iPSCs through the utilization of base editing technology.