論文 - 詳細
| RRC ID | 70185 |
|---|---|
| 著者 | Seleit A, Aulehla A, Paix A. |
| タイトル | Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach. |
| ジャーナル | Elife |
| Abstract |
The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter. |
| 巻・号 | 10 |
| 公開日 | 2021-12-6 |
| DOI | 10.7554/eLife.75050 |
| PII | 75050 |
| PMID | 34870593 |
| PMC | PMC8691840 |
| MeSH | Animals CRISPR-Cas Systems / genetics* Developmental Biology Gene Editing / methods* Gene Knock-In Techniques Oryzias / genetics* Oryzias / physiology* Protein Transport / genetics* Protein Transport / physiology* |
| IF | 7.08 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 24 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| メダカ | TG1421 Tg(cdh2-eGFP) TG1422 Tg(eGFP-cbx1b) TG1423 Tg(eGFP-rab11a) TG1424 Tg(mapre1b-Linker-3xFlag-mScarlet) TG1425 Tg(mNeonGreen-HAtag-Linker-myosinh) TG1426 Tg(mScarlet-pcna) |