Deciphering the Design Rules of Toehold-Gated sgRNA for Conditional Activation of Gene Expression and Protein Degradation in Mammalian Cells

Abstract

A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells

“…S4A, ESI †), a thgRNA design with the best signal to background ratio in HeLa cells. 13 A synthetic trigger was placed behind the miR-gated sgRNA and is released by a sandwiching hammerhead (HH) and hepatitis delta virus (HDV) ribozyme (Fig. S3, ESI †).…”

“…S5A, ESI †). 13 Since the toehold sequence has been shown to interact with the loop region, thgNT-F was designed specifically with a short 5-nt toehold region to minimize background leak. Because RISC complex cleavage results in 11nt fragment remaining on both 5 0 and 3 0 ends, 25 we suspect that the extra 11nt flanking sequence to the 5nt toehold led to increased non-specific interaction with the loop.…”

“…The higher knockdown level is consistent with the improved degradation efficiency of the Nsa5-IpaH9.8 ubiquibody. 13 In this work, we have successfully created thgRNAs that are responsive to endogenous mRNAs in mammalian cells. We showed that thgRNAs expressed from the hU6 promoter cannot interact efficiently with rapidly exported full-length mRNAs in the nucleus.…”

“…Our lab has recently demonstrated that the level of activation is 3-fold lower for RNA triggers expressed using the CMV promoter versus the hU6 promoter. 13 This result indicates that the rapid translocation of RNA triggers expressed using the type II polymerase reduces duration of strand displacement within the nucleus for activation. This limitation renders full-length mRNA ineffective as a trigger for activation.…”

“…We have designed thgRNA, g2v2thg-F, 13 containing a 5nt toehold region, 15nt loop, and a fully hybridized stem region that is activated by a specific region of the mCherry mRNA. A short synthetic trigger was shown to activate GFP expression from a minimum CMV promoter by 15-fold based on CRISPRa.…”