RNA targeting with CRISPR-Cas 13 a

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 Correspondence should be addressed to F.Z. (zhang@broadinstitute.org). *These authors contributed equally to this work. Author Contributions: O.O.A, J.S.G, and F.Z. conceived and designed the study. O.O.A, J.S.G, P.E., J.J., and V.V. performed experiments. O.O.A and J.S.G analyzed data. S.H. performed select microscopy experiments. J.J.B. performed the plant protoplasts knockdown experiments. O.O.A, J.S.G, E.S.L, and F.Z. wrote the paper with input from D.F.V and A.T. and help from all authors. Code availability: Relevant code for data analysis is available on Github (https://github.com/fengzhanglab) Data availability: High throughput sequencing data related to this study is available at BioProject PRJNA383832. HHS Public Access Author manuscript Nature. Author manuscript; available in PMC 2018 April 12. Published in final edited form as: Nature. 2017 October 12; 550(7675): 280–284. doi:10.1038/nature24049. A uhor M anscript