Building Endogenous Gene Connections through RNA Self-Assembly Controlled CRISPR/Cas9 Function

Abstract

Construction of synthetic circuits that can artificially establish endogenous gene connections is essential to introduce new phenotypes for cellular behaviors. Given the diversity of endogenous genes, it lacks a general and easy-to-design toolbox to manipulate the genetic network. Here we present a type of self-assembly-induced RNA circuit that can directly build regulatory connections between endogenous genes. Inspired from the natural assembling process of guide RNA in the CRISPR/Cas9 complex, this design employs an independent trigger RNA strand to induce the formation of a ternary guide RNA assembly for functional control of CRISPR/Cas9. With this general principle, expressional regulations of endogenous genes can be controlled by totally independent endogenous small RNAs and mRNAs in <i>E. coli</i> via activatable CRISPR/Cas9 function. Moreover, the cellular phenotype of <i>E. coli</i> is successfully programmed with introduction of new gene connections. In addition, the functionality of this design is also verified in the mammalian system. This self-assembly-based RNA circuit exhibits a great flexibility and simplicity of design and provides a unique approach to build endogenous gene connections, which paves a broad way toward manipulation of cellular genetic networks.