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Abudayyeh OO
; Gootenberg JS
; Essletzbichler P
; Han S
; Joung J
; Belanto JJ
; Verdine V
; Cox DBT
; Kellner MJ
; Regev A
; Lander ES
; Voytas DF
; Ting AY
; Zhang F
Nature
2017[Oct]; 550
(7675
): 280-284
PMID28976959
show ga
RNA has important and diverse roles in biology, but molecular tools to manipulate
and measure it are limited. For example, RNA interference can efficiently
knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs
typically relies on the introduction of exogenous tags. Here we demonstrate that
the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a
(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.
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