Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan.
Graduate School of Medicine, Kyoto University, Kyoto, Japan.
CRISPR J. 2024 Oct;7(5):293-304. doi: 10.1089/crispr.2024.0028.
Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the benchmark locus and at the Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.
利用可编程核酸酶对人诱导多能干细胞 (iPS) 进行基因编辑可促进可靠的疾病模型的建立，但利用双链断裂修复的方法往往会产生随机的脱靶副产物。Prime editing (PE) 将 Cas9 切口酶与逆转录酶和包含修复模板的 PE 指导 RNA (pegRNA) 结合使用，以减少副产物。我们实施了一种符合 GMP 标准的转染 Cas9 或 PE-2A-mCherry 质粒的方案，根据 PE 表达水平来跟踪和分离人类 iPS 细胞。我们在 GFP 到 BFP 的转换测定中比较了 Cas9 和基于 PE 的方法的编辑结果，在基准基因座和阿尔茨海默病风险基因座，发现 PE 在高表达水平下具有更高的精确性。此外，对 PE 表达水平进行分选影响了靶基因座的等位基因编辑结果。我们期望我们的发现将有助于创建具有有意杂合和纯合基因型的基因编辑人类 iPS 细胞。
