Fluorescent Guide RNAs for Understanding Grnas Expressed from Pol II Promoters

Introduction: CRISPR technology has quickly become an important tool in the synthetic biology repertoire, used for applications such as induced mutation and gene silencing, genome labeling, and activation/repression. The relatively straight-forward design principles of the gRNAs, together with the ease with which gRNAs or Cas9 proteins can be modified to recruit or contain other active domains, promises to simplify and expand the field of de novo genetic circuits and allow researchers convenient ways to interact with existing networks. This shows great potential for bioproduction and therapeutic applications, either rewiring faulty genetic networks or normalizing unbalanced systems. However, most systems of interest are composed primarily of RNA Polymerase II (Pol II) driven components, while non-coding gRNAs are most commonly produced by RNA Polymerase III (Pol III). While several methods exist to convert the mRNA products of Pol II promoters into functional gRNAs¹, none of these methods produce gRNA capable of inducing downstream effects as powerfully as those produced from a strong Pol III promoter. In this study, we aim to increase the efficiency of Pol II gRNA production in order to allow researchers to interface more dynamically with existing gene networks.

Materials and Methods: Plasmids were made containing either Pol III or inducible Pol II promoters driving gRNAs containing the fluorescent RNA aptamer Broccoli². Pol II constructs included either ribozymes or Csy4 sites, to allow mRNA editing into a functional gRNA. These basic constructs were then modified to increase their effectiveness in producing gRNAs: modified terminators, intronic additions, multiplexing, and an RNA nuclear localization sequence. Simple repressor circuits, in which the gRNA repressed the expression of infrared fluorescent protein (iRFP) were transfected into human HEK293rtTA cells and analyzed via flow cytometry. Fluorescence increase of the Broccoli aptamer was compared to the decrease of the iRFP signal to determine effectiveness of gRNA repression.

Results and Discussion: Initial data indicates that the gRNA transcript can be modified to fluoresce without affecting its function, and that mRNA can be edited into functional fluorescent guides through multiple methods. Further, we have demonstrated that that Pol II driven repression efficiency can be increased by multiplexing several repeats of the gRNA together, as well as by changing the gene’s terminator to one containing a minimal polyA sequence, thereby reducing nuclear export.

Conclusions: This work shows that gRNA expression from Pol II promoters can be improved through several different methods. They can be used in tandem or employed individually for niche situations. One limitation of all of these methods, however, is the efficiency of the editors which cleave mRNA into functional gRNAs. If this can be raised, overall efficiency will be improved, so one potential extension of this work would be a more thorough comparison of mRNA editors and modifications to improve their function.

References:

1 Nissim, L. Cell. vol. 54, 698-710, 2014.

2 Filonov, G.S., Jour. of Amer. Chem. Soc. vol. 146, 16299-16308, 2014.