Journal Article

RNA targeting with CRISPR–Cas13

Voytas, Daniel F., Essletzbichler, Patrick, Han, Shuo, Lander, Eric S., Regev, Aviv, Gootenberg, Jonathan S., Verdine, Vanessa, Kellner, Max J., Ting, Alice Y., Joung, Julia, Belanto, Joseph J., Abudayyeh, Omar O., Cox, David B. T., Zhang, Feng

2017

Overview

The class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13 can be engineered for RNA knockdown and binding, expanding the CRISPR toolset with a flexible platform for studying RNA in mammalian cells and therapeutic development. A CRISPR way to knockdown RNA CRISPR–Cas prokaryotic defence systems have provided versatile tools for DNA editing. Here, the authors demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13a (previously known as C2c2) can be engineered for RNA knockdown and binding in mammalian cells. This addition to the CRISPR toolbox expands its potential uses to transcript tracking and knockdown. RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference 1 , 2 , 3 can efficiently knockdown RNAs, but it is prone to off-target effects 4 , and visualizing RNAs typically relies on the introduction of exogenous tags 5 . Here we demonstrate that the class 2 type VI 6 , 7 RNA-guided RNA-targeting CRISPR–Cas effector Cas13a 8 (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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 13 / 14 / 38/35 / 38/39 / 38/91 / 42/109 / 42/44 / 631/1647/1511 / 631/208/212/748 / 631/61/338/552 / 631/61/391 / Binding / Biocatalysis / Cell Line, Tumor / Cell Survival / CRISPR / CRISPR-Associated Proteins - chemistry / CRISPR-Associated Proteins - genetics / CRISPR-Associated Proteins - metabolism / CRISPR-Cas Systems / E coli / Escherichia coli - genetics / Gene Editing / Gene expression / Gene Knockdown Techniques - methods / Genes, Reporter - genetics / Genetic engineering / Genomes / Genomics / HEK293 Cells / Humanities and Social Sciences / Humans / Interference / Leptotrichia - enzymology / Leptotrichia - genetics / letter / Mammalian cells / Mammals / multidisciplinary / Oryza sativa / Panicum virgatum / Plant cells / Plant Cells - metabolism / Ribonucleic acid / RNA / RNA - analysis / RNA - genetics / RNA - metabolism / RNA Interference / RNA polymerase / RNA-mediated interference / Science / Stress, Physiological / Substrate Specificity