Ryo Niwa, Tomoko Matsumoto, Alexander Y Liu, Maki Kawato, Takayuki Kondo, Kayoko Tsukita, Peter Gee, Haruhisa Inoue, Thomas L Maurissen, Knut Woltjen
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引用次数: 0
摘要
使用可编程核酸酶对人类诱导多能干细胞(iPS)进行基因编辑有助于建立可靠的疾病模型,但使用双链断裂修复的方法往往会产生随机的靶上副产物。Prime editing(PE)将Cas9缺口酶与反转录酶和编码修复模板的PE引导RNA(pegRNA)结合起来,以减少副产物。我们采用与 GMP 兼容的方案转染 Cas9- 或 PE-2A-mCherry 质粒,根据 PE 表达水平跟踪和分馏人类 iPS 细胞。在 HEK3 基准基因座和 APOE 阿尔茨海默氏症风险基因座的 GFP 到 BFP 转换试验中,我们比较了 Cas9 和 PE 方法的编辑结果,结果显示 PE 在高表达水平下的精确度更高。此外,根据 PE 表达水平对细胞进行分选会影响目标基因座的等位基因编辑结果。我们希望我们的发现将有助于创建具有有意杂合和同源基因型的基因编辑人类 iPS 细胞。
Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.
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 HEK3 benchmark locus and at the APOE 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.
CRISPR JournalBiochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍:
In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR.
Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.