Fine-Tuning Site-Directed RNA Editing: Development of Inducible gRNA Expression Systems

Site-directed RNA editing has emerged as an alternative for genome editing and has advanced greatly in recent years. Many systems have been developed by exploiting deaminases which naturally edit transcripts in eukaryotes. These systems rely on endogenous or engineered versions of adenosine deaminase acting on RNA (ADAR) or APOBECs. ADARs catalyze the determination of adenosines to inosine (A-to-I), which is then recognized by the translation machinery as guanine. APOBECs catalyze deamination of cytosine to uracil (C-to-U) on RNA or DNA. RNA editing tools such as LEAPER, where a 70-150 base long gRNA containing a cytosine (C) mismatch opposite the target adenosine (A) is used for recruiting endogenous ADARs without the need of exogenous enzymes. On the other hand, tools like dDiCas7-11-hADAR2DD, REPAIR and CURE rely on engineered versions of ADAR or APOBEC3A fused to a catalytically dead Cas subunits. However, for all these systems the key component facilitating RNA editing is the gRNA. When any of these systems are provided through plasmidic vectors, either by transfection or viral transduction, gRNAs are placed under the control of the U6 promoter. The U6 promoter has been extensively used for expression of small RNAs in a variety of techniques, short hairpin RNAs, CRISPR gRNAs, RNAi etc.. While this approach allows efficient synthesis and limits the number of components required, it is nonetheless difficult to modulate. Moreover, it might not represent the best option if the gRNAs are meant to reach the cytosol, as in the case of programmable RNA editing. To overcome this challenge, I have devised a system by which the gRNAs are synthesized in the cytosol via transcription mediated by T7 RNA polymerase (RNAP). For this, I have placed the gRNAs are under the control of a T7 promoter, coupled with a modified T7 termination sequence to prevent run-away transcription. I then use a plasmid encoding for a mammalian cell optimized T7 RNAP. Through co-transfection of all components together with a dual-fluorescent reporter system, I show efficient editing efficiency of the reporter construct using LEAPER. My system was able to perform as efficiently as the U6 promoter driven gRNA expression, with the added advantage of control over gRNA expression without any leakage. I further optimized conditions of the systems to achieve improved editing levels and tested my system on in-house generated ADAR1, ADAR2 and ADAR double knock-out HEK293-T cells. With development of improved SDRE tools I tested editing efficiencies of circular gRNAs. Based on this I implemented the T7 RNAP system on circular gRNAs using the LEAPER 2.0 design, once more achieving comparable editing levels to the U6 driven version. Building upon the T7 RNAP mediated system, I further developed the system by placing T7 RNAP expression under the control of a TET-ON operon. I also attempted to develop cell specific expression systems using OCT4 and SOX2 promoters and are currently testing these on relevant cell types. In conclusion, I have successfully developed and validated a novel T7 RNAP-mediated system for controlled expression of SDRE gRNAs. Offering a tunable alternative to the conventional U6-driven systems. Furthermore, the work demonstrates the adaptability of this approach to various gRNA designs, including circular RNAs, and its potential for cell-type specific applications through the implementation of inducible and cell-specific promoters. These advancements pave the way for more controlled and targeted RNA editing strategies in diverse biological contexts.