High-Performance Allosteric Conditional Guide RNAs for Mammalian Cell-Selective Regulation of CRISPR/Cas

The activity of a conditional guide RNA (cgRNA) is dependent on the presence or absence of an RNA trigger, enabling cell-selective regulation of CRISPR/Cas function. cgRNAs are programmable at two levels, with the target-binding sequence controlling the target of Cas activity (edit, silence, or induce a gene of choice) and the trigger-binding sequence controlling the scope of Cas activity (subset of cells expressing the trigger RNA). Allosteric cgRNA mechanisms enable independent design of the target and trigger sequences, providing the flexibility to select the regulatory target and scope independently. Building on prior advances in dynamic RNA nanotechnology that demonstrated the cgRNA concept, here we set the goal of engineering high-performance allosteric cgRNA mechanisms for the mammalian setting, pursuing both ON → OFF logic (conditional inactivation by an RNA trigger) and OFF → ON logic (conditional activation by an RNA trigger). For each mechanism, libraries of orthogonal cgRNA/trigger pairs were designed using NUPACK. In HEK 293T cells expressing cgRNAs, triggers, and inducing dCas9: (1) a library of four ON → OFF "terminator switch" cgRNAs exhibit a median fold-change of ≈50×, a median fractional dynamic range of ≈20%, and a median crosstalk modulus of ≈9%; (2) a library of three OFF → ON "split-terminator switch" cgRNAs exhibit a median fold-change of ≈150×, a median fractional dynamic range of ≈50%, and a median crosstalk modulus of ≈4%. Further, we demonstrate that xrRNA elements that protect viral RNAs from degradation by exoribonucleases can dramatically enhance the performance of RNA synthetic biology. The high-performance allosteric cgRNAs demonstrated here for ON → OFF and OFF → ON logic in mammalian cells provide a foundation for pursuing applications of programmable cell-selective regulation.