A CRISPR-del-based pipeline for complete gene knockout in human diploid cells
| العنوان: | A CRISPR-del-based pipeline for complete gene knockout in human diploid cells |
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| المؤلفون: | Takuma Komori, Shoji Hata, Akira Mabuchi, Mariya Genova, Tomoki Harada, Masamitsu Fukuyama, Takumi Chinen, Daiju Kitagawa |
| المصدر: | Journal of Cell Science. 136 |
| بيانات النشر: | The Company of Biologists, 2023. |
| سنة النشر: | 2023 |
| مصطلحات موضوعية: | Cell culture, Cas9, CRISPR, Computational biology, Cell Biology, Biology, Ploidy, Indel, Gene, Gene knockout, Chromosomal Deletion |
| الوصف: | The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel-based gene disruption. In this study, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform “complete” gene knockout efficiently in non-transformed human diploid RPE1 cells. By optimizing several procedures, the CRISPR-del pipeline allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enables the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is a high-throughput approach for performing “complete” gene knockout in RPE1 cells. |
| تدمد: | 1477-9137 0021-9533 |
| DOI: | 10.1242/jcs.260000 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c5686ac41fed02c879fe9a81d261a05d https://doi.org/10.1242/jcs.260000 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....c5686ac41fed02c879fe9a81d261a05d |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14779137 00219533 |
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| DOI: | 10.1242/jcs.260000 |