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.

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

Signal Transduction in Human Cell Lysate via Dynamic RNA Nanotechnology.

Dynamic RNA nanotechnology with small conditional RNAs (scRNAs) offers a promising conceptual approach to introducing synthetic regulatory links into endogenous biological circuits. Here, we use human cell lysate containing functional Dicer and RNases as a testbed for engineering scRNAs for conditional RNA interference (RNAi). scRNAs perform signal transduction via conditional shape change: detection of a subsequence of mRNA input X triggers formation of a Dicer substrate that is processed to yield small interfering RNA (siRNA) output anti-Y targeting independent mRNA Y for destruction. Automated sequence design is performed using the reaction pathway designer within NUPACK to encode this conditional hybridization cascade into the scRNA sequence subject to the sequence constraints imposed by X and Y. Because it is difficult for secondary structure models to predict which subsequences of mRNA input X will be accessible for detection, here we develop the RNAhyb method to experimentally determine accessible windows within the mRNA that are provided to the designer as sequence constraints. We demonstrate the programmability of scRNA regulators by engineering scRNAs for transducing in both directions between two full-length mRNAs X and Y, corresponding to either the forward molecular logic "if X then not Y" (X [Formula: see text] Y) or the reverse molecular logic "if Y then not X" (Y [Formula: see text] X). In human cell lysate, we observe a strong OFF/ON conditional response with low crosstalk, corresponding to a ≈20-fold increase in production of the siRNA output in response to the cognate versus noncognate full-length mRNA input. 2'OMe-RNA chemical modifications protect signal transduction reactants and intermediates against RNase degradation while enabling Dicer processing of signal transduction products. Because diverse biological pathways interact with RNA, scRNAs that transduce between detection of endogenous RNA inputs and production of biologically active RNA outputs hold great promise as a synthetic regulatory paradigm.

Conditional Dicer substrate formation via shape and sequence transduction with small conditional RNAs.

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate 'conditional RNAi' corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA 'detection target' X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA 'silencing target' Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly.