Current strategies for Site-Directed RNA Editing using ADARs

Abstract

Adenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A's) to inosines (I's) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I's base pair like guanosines (G's). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering protein sequence and function. This ability to re-code makes ADAR an attractive therapeutic tool to correct genetic mutations within mRNA. The main challenge in doing so is to re-direct ADAR's catalytic activity towards A's that are not naturally edited, a process termed Site-Directed RNA Editing (SDRE). Recently, a handful of labs have taken up this challenge and two basic strategies have emerged. The first involves redirecting endogenous ADAR to new sites by making editable structures using antisense RNA oligonucleotides. The second also utilizes antisense RNA oligonucleotides, but it uses them as guides to deliver the catalytic domain of engineered ADARs to new sites, much as CRISPR guides deliver Cas nucleases. In fact, despite the intense current focus on CRISPR-Cas9 genome editing, SDRE offers a number of distinct advantages. In the present review we will discuss these strategies in greater detail, focusing on the concepts on which they are based, how they were developed and tested, and their respective advantages and disadvantages. Though the precise and efficient re-direction of ADAR activity still remains a challenge, the systems that are being developed lay the foundation for SDRE as a powerful tool for transient genome editing.

Keywords: ADAR; Antisense oligo; Guide RNA; Off-target events; RNA editing.

Figure 1.. Recruiting endogenous ADAR.

Endogenous ADAR1 or 2 is recruited by mimicking native RNA structures. Antisense RNA oligo containing a C mismatch is composed mainly of two parts: 3’ region complimentary to the target mRNA (in green) and a 5’ region that forms the secondary structure resembling the endogenous GluR2 R/G target sequence (in yellow). This structure is recognized by ADAR’s dsRBM (in orange) that guide the DD (in red) to the target A under a C mismatch.

Figure 2.. SNAP-tag Strategy.

The SNAP-tag enzyme (in orange) covalently binds to the gRNA (in green) through a linker region (in magenta). The gRNA then brings the DD (in red) to a specific region of a message and also creates a suitable dsRNA substrate surrounding the targeted A. The gRNA contains a C mismatch at the target A to promote more efficient editing.

Figure 3.. λN-BoxB strategy.

The editing enzyme containing the λN peptides (λNx4, in orange) fused to the ADAR2 deaminase domain (in red) is guided to the target mRNA by the gRNA (in green). This gRNA is composed of three regions: i) a central region fully complementary to the target mRNA except for a C mismatch at the target A, ii) two BoxB RNA hairpins positioned strategically to promote efficient editing and, iii) two fully complementary regions on each terminus of the gRNA to stabilize it.

Figure 4.. MS2 strategy.

The gRNA consists of two parts: The 5’ region fully complementary to the target mRNA (in purple). At the target A, there is C mismatch to favor editing, while a G mismatch is used to reduce editing in non-target As. The 3’ region consists of a 6X tandem RNA stem-AUCA loop repetition that is recognized by the MS2 protein (in orange) fused to the DD of human ADAR1 (in red).

Figure 5.. CRISPR-Cas13b Strategy.

The gRNA (in green) is composed of a 50 nt long 5’ region complementary to the target sequence, with a C mismatch opposite to the target A. The 3’ region of the gRNA is a 36 nt hairpin-forming the Direct Repeat recognized by Cas13b. Catalytically dead Cas13b (DCas13b in orange) fused to the DD of human ADAR1 or 2 (in red) binds to the Direct Repeat hairpin and positions the DD of ADAR1/2 in the vicinity of the target A on a mRNA (in purple).