RNA targeting with CRISPR–Cas13
| Autor: | Patrick Essletzbichler, Alice Y. Ting, David Benjamin Turitz Cox, Omar O. Abudayyeh, Aviv Regev, Joseph J. Belanto, Eric S. Lander, Max J. Kellner, Daniel F. Voytas, Feng Zhang, Julia Joung, Jonathan S. Gootenberg, Vanessa Verdine, Shuo Han |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Small interfering RNA Cell Survival CRISPR-Associated Proteins Biology Article Substrate Specificity 03 medical and health sciences 0302 clinical medicine Genes Reporter Stress Physiological DNA-directed RNA interference RNA interference Cell Line Tumor Plant Cells Sense (molecular biology) Escherichia coli Humans Leptotrichia Gene Editing Genetics Gene knockdown Multidisciplinary RNA Cell biology RNA silencing HEK293 Cells 030104 developmental biology RNA editing Gene Knockdown Techniques 030220 oncology & carcinogenesis Biocatalysis RNA Interference CRISPR-Cas Systems |
| Zdroj: | Nature. 550:280-284 |
| ISSN: | 1476-4687 0028-0836 |
| DOI: | 10.1038/nature24049 |
| Popis: | RNA plays important and diverse roles in biology, but molecular tools to manipulate and measure RNA are limited. For example, RNA interference (RNAi)1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here, we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8 (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of fifteen orthologs in E. coli, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts. We demonstrate that LwaCas13a is capable of providing comparable levels of knockdown as RNAi, but with dramatically improved specificity. Moreover, catalytically inactive LwaCas13a maintains targeted RNA binding, allowing for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for RNA targeting with wide applicability for studying RNA in mammalian cells. |
| Databáze: | OpenAIRE |
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