論文 - 詳細
| RRC ID | 35757 |
|---|---|
| 著者 | Yoshimi K, Kunihiro Y, Kaneko T, Nagahora H, Voigt B, Mashimo T. |
| タイトル | ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. |
| ジャーナル | Nat Commun |
| Abstract |
The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms. |
| 巻・号 | 7 |
| ページ | 10431 |
| 公開日 | 2016-1-20 |
| DOI | 10.1038/ncomms10431 |
| PII | ncomms10431 |
| PMID | 26786405 |
| PMC | PMC4736110 |
| MeSH | Animals Antigens, Differentiation / genetics CRISPR-Cas Systems / genetics* Clustered Regularly Interspaced Short Palindromic Repeats / genetics Female Gene Knock-In Techniques Genetic Engineering / methods* Homologous Recombination / genetics Humans Male Mice Oligodeoxyribonucleotides / genetics* Rats Receptors, Immunologic / genetics Zygote / metabolism* |
| IF | 12.121 |
| WOS 分野 | BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 81 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 6.0 |
| リソース情報 | |
| 遺伝子材料 | T7-NLS hCas9-pA (RDB13130) pCAGGS (RDB08938) |