Design of Conditional Guide RNAs for the Logical Regulation of Gene Expression

The CRISPR interference (CRISPRi) is an RNA-guided regulator that silences gene expression by binding to its cognate DNA target, halting transcription in both prokaryotic and eukaryotic cells. Recent advances in RNA synthetic biology have endowed CRISPR guide RNAs (gRNAs) with conditional functionality: these so-called conditional guide RNAs (cgRNAs) fold into strong hairpins that block their activity until a specific trigger RNA is present. Upon introduction of the cognate trigger RNAs, the hairpin structure unfolds, allowing the activated cgRNA to direct transcriptional repression with large dynamic ranges, minimal crosstalk, expanded tunability, and logic-gated signal processing. Furthermore, cgRNAs can be integrated into endogenous gene circuits to achieve sophisticated and logical regulation of gene expression. This chapter describes the design of cgRNAs and provides detailed protocols for their in vivo characterization in E. coli.