Logical regulation of endogenous gene expression using programmable, multi-input processing CRISPR guide RNAs

The CRISPR-Cas system provides a versatile RNA-guided approach for a broad range of applications. Thanks to advances in RNA synthetic biology, the engineering of guide RNAs (gRNAs) has enabled the conditional control of the CRISPR-Cas system. However, achieving precise regulation of the CRISPR-Cas system for efficient modulation of internal metabolic processes remains challenging. In this work, we developed a robust dCas9 regulator with engineered conditional gRNAs to enable tight control of endogenous genes. Our conditional gRNAs in Escherichia coli can control gene expression upon specific interaction with trigger RNAs with a dynamic range as high as 130-fold, evaluating up to a three-input logic A OR (B AND C). The conditional gRNA-mediated targeting of endogenous metabolic genes, lacZ, malT and poxB, caused differential regulation of growth in Escherichia coli via metabolic flux control. Further, conditional gRNAs could regulate essential cytoskeleton genes, ftsZ and mreB, to control cell filamentation and division. Finally, three types of two-input logic gates could be applied for the conditional control of ftsZ regulation, resulting in morphological changes. The successful operation and application of conditional gRNAs based on programmable RNA interactions suggests that our system could be compatible with other Cas-effectors and implemented in other host organisms.