論文 - 詳細
| RRC ID | 81897 |
|---|---|
| 著者 | Niwa R, Matsumoto T, Liu AY, Kawato M, Kondo T, Tsukita K, Gee P, Inoue H, Maurissen TL, Woltjen K. |
| タイトル | Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells. |
| ジャーナル | CRISPR J |
| Abstract |
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. |
| 巻・号 | 7(5) |
| ページ | 293-304 |
| 公開日 | 2024-10-1 |
| DOI | 10.1089/crispr.2024.0028 |
| PMID | 39436282 |
| MeSH | Alleles* Alzheimer Disease / genetics Alzheimer Disease / therapy Apolipoproteins E / genetics CRISPR-Associated Protein 9 / genetics CRISPR-Associated Protein 9 / metabolism CRISPR-Cas Systems* Gene Editing* / methods Humans Induced Pluripotent Stem Cells* / metabolism RNA, Guide, CRISPR-Cas Systems* / genetics |
| リソース情報 | |
| ヒト・動物細胞 | 409B2(HPS0076) |