Exploiting the endogenous: An ADAR way to edit RNA by only adding oligos

Genome editing, with its permanent modification of DNA, necessitates that the editing is conducted both safely and efficiently. An alternative mechanism, that could be both reversible and dose dependent in terms of percentage editing, is to edit at the RNA stage. However, this precise RNA editing faces similar safety and efficiency requirements if it is to have therapeutic potential. Previously, RNA editing could only be achieved if the RNA editing proteins, such as ADAR, were overexpressed. However, ADAR is naturally expressed in cells and so an alternative and potentially safer and easier method would be to exploit this endogenous expression instead. Terming the technique “RESTORE”, a recent Nature Biotechnology paper 1 by Merkle et al., have achieved just that.

restore

Adenosine deaminase acting on RNA (ADAR)

ADAR proteins are enzymes involved in the conversion of adenosine (A) to inosine (I) in RNA. Inosine is a non-conventional nucleoside found in RNA and so is read as guanine (G) by the cell. This effective A to G conversion in RNA is a type of RNA editing. Editing the sequence in mRNA can alter the amino acids that is codes for or add a stop codon. Changes in the non-coding parts of mRNA can also have an effect by altering the binding ability of a plethora of RNA binding proteins and thus the degradation rate, localisation and translation rate can be modified. Importantly, ADAR achieves its editing capability by binding double stranded RNA. But RNA is single stranded – how can ADAR then be recruited?

The principle of site-specific RNA editing with ADAR

There are two key steps that are required for RNA editing by ADAR to work. Firstly, the target RNA and location on that RNA to be edited needs to be recognised. Then, ADAR needs to be recruited so that it can carry out the deamination of adenosine (A) to inosine (I). Both these steps can be achieved through the design and engineering of a single RNA oligonucleotide. This RNA consists of two domains; a specificity domain that is the antisense of the target RNA and an ADAR recruiting domain which forms an imperfect double-stranded structure. These RNAs that guide editing are referred to as antisense oligonucleotides (ASOs).

aso

Merkle’s team designed numerous ASOs during this paper to find a design that optimised ADAR recruitment and RNA editing. This involved changing the length of the specificity domain, adding modifications to the RNA to increase its stability and half-life in the cell, or enabling the ASO to bind to RNA more strongly. There are different types of ADAR that are expressed endogenously in a cell – the team found that their ASO recruited an ADAR1 isoform best. ADAR1 expression can be increased in the presence of interferon (IFN-α) – notably, percentage RNA editing was greater when IFN-α was applied to the cells. The levels of ADAR in the cell were also sufficient to enable more than one RNA site to be modified simultaneously.

After designing and testing that their ASOs worked, the team termed the process RESTORE, for recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing – a tad obscure, but I like it!

Exploiting endogenous ADAR –> Minimal side-effects

The benefit of using endogenously expressed ADAR is that no exogenous overexpression of ADAR is required. Not only would this have the problem of genetically manipulating cells to overexpress ADAR, but these non-physiological levels of ADAR are more likely to interfere with natural levels of RNA editing within a cell. However, whether this would be a problem for RESTORE needed to be checked. Deep RNA sequencing was performed whilst using an ASO to target and edit GAPDH mRNA – 14/16,716 off-target sites were found to be differently edited when IFN-α was also added, showing that there is only a minimal off-target impact. This is good to know for example when using RESTORE as a tool to study RNA function in a cell. To get a more accurate understanding, the tool needs to have minimal disruption to the cell.

RESTORE to restore defective cells?

Merkle’s team show RESTORE to be an effective RNA editing strategy that could potentially be used to target many mRNAs simultaneously. Not only does RESTORE have a low off-target impact, but it provides a mechanism that could be extrapolated to therapeutic purposes due the simplicity in only having to add the ASO. Whilst simplicity is probably not quite the correct word, since even targeting ASOs to target cells is no trivial task, there is evidence that this has been achieved in liver cells. So, there is definitely potential! However, whilst the efficacy of editing can be seen from the sequencing data, it would be interesting to see the phenotypic efficacy that editing of only a percentage of RNA in a cell can have. Also, does the presence of inosine make the RNA unstable? Another limitation is that with this method only A to G editing can be performed. Nonetheless, the design of RESTORE in addition to the experimental design used in this paper provide a strong framework on which to further adapt this method to try and recruit alternative RNA editing proteins that could expand the editing capacity.

Further Reading

  1. Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019).

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