| Autor: |
Joseph M Replogle, Jessica L Bonnar, Angela N Pogson, Christina R Liem, Nolan K Maier, Yufang Ding, Baylee J Russell, Xingren Wang, Kun Leng, Alina Guna, Thomas M Norman, Ryan A Pak, Daniel M Ramos, Michael E Ward, Luke A Gilbert, Martin Kampmann, Jonathan S Weissman, Marco Jost |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
|
| Zdroj: |
eLife, Vol 11 (2022) |
| Druh dokumentu: |
article |
| ISSN: |
2050-084X |
| DOI: |
10.7554/eLife.81856 |
| Popis: |
CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1–3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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