Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs

Abstract

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remains low using current guide RNA designs. We engineered c ircular AD AR recruiting guide RNAs (cadRNAs) to enable more efficient programmable A-to-I RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications,

ADAR2 enzymes: efficient site-specific RNA editors with gene therapy aspirations

“…Some early approaches to retargeting ADAR RNA editing used fusions of other RNAbinding proteins to an ADAR deaminase domain and some of these gave efficient target editing; however, they also identified a critical problem arising from high off-target editing (Montiel-Gonzalez et al 2019). This, and difficulties expected with expression of proteins and possible immunogenicity of foreign proteins in humans mean that current approaches concentrate on retargeting endogenous ADAR proteins by providing an ADAR-recruiting 'guide' RNA that will also base pair across the mutation site and make the target adenosine available for editing (Katrekar et al 2022;Reautschnig et al 2022;Yi et al 2022). Studies on ADAR2 have been the predominant source of information on how ADARs recognize RNA substrates for highly efficient site-specific RNA editing and on how we might design guide RNAs to retarget ADARs for gene therapy.…”

Programmable RNA Sensing for Cell Monitoring and Manipulation

“…Improved readrRNA stability and efRNA translation, e.g. by using circular 46,47 or chemical modified READR RNAs 24,48 , should boost its efficacy. Innovation in nucleic acid delivery, such as liposome nanoparticles 49 , SEND (selective endogenous encapsidation for cellular delivery) system 29 , and viral capsid proteins that steer tissue and species tropism 50 , will enhance the ease and range of CellREADR applications.…”

Circular RNA and Its Roles in the Occurrence, Development, Diagnosis of Cancer

“…Katrekar et al. engineered a highly stable circular ADAR-recruiting guide RNA (cadRNA) to recruit endogenous ADARs, improving the efficiency and durability of RNA editing ( 26 ). The engineered circ-arRNAs designed by Yi et al.’s LEAPER2.0 system have much higher editing efficiency than the corresponding linear arRNAs, which greatly improves the efficiency and robustness of RNA editing ( 27 ).…”