Abstract

The Cas9 enzyme is a programmable endonuclease, whose target sequence is directed by a companion RNA guide. Cas9 and RNA guides have revolutionized biology, enabling facile editing of the genome in almost all organisms. Controlling where and when Cas9 and the guide operate is indispensable for many fields ranging from developmental biology to therapeutics, but it remains a challenge. Most methods focus on controlling Cas9 with physico-chemical means (which lack finesse, precision or multiplexing), or transcriptional tools (which are slow and difficult to design). Rather than directly engineering Cas9, engineering the RNA guide itself has emerged as a more general and potent way to manage the activity of Cas9. Here we report smart RNA guides that are conditionally activated by the presence of a specific RNA opener. Contrary to most previous approaches, the design affords ample freedom as spacer and the opener are independent. We demonstrate this flexibility by operating SmartGuides activated by a panel of miRNA relevant for human health, and by composing SmartGuides in Boolean logic circuits. Lastly, we test the SmartGuides in mammalian cells - validating the basics tenets of the design, but also highlighting the challenges that remain to be lifted for in-vivo operation.