RNA targeting with CRISPR–Cas13
Author(s)Essletzbichler, Patrick; Belanto, Joseph J.; Verdine, Vanessa; Kellner, Max J.; Voytas, Daniel F.; Abudayyeh, Omar Osama; Gootenberg, Jonathan S; Han, Shuo; Joung, Julia; Cox, David Benjamin Turitz; Regev, Aviv; Lander, Eric Steven; Ting, Alice Y; Zhang, Feng; ... Show more Show less
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RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1-3 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 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
DepartmentHarvard University--MIT Division of Health Sciences and Technology; Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences; Massachusetts Institute of Technology. Department of Chemistry; McGovern Institute for Brain Research at MIT
Nature Publishing Group
Abudayyeh, Omar O. et al. “RNA Targeting with CRISPR–Cas13.” Nature 550, 7675 (October 2017): 280–284 © 2017 Macmillan Publishers Limited, part of Springer Nature
Author's final manuscript