CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing.

Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.

Site-directed RNA editing might provide a safer or more effective alternative to genome editing in certain clinical scenarios. Until now, RNA editing has relied on overexpression of exogenous RNA editing enzymes or of endogenous human ADAR (adenosine deaminase acting on RNA) enzymes. Here we describe the engineering of chemically optimized antisense oligonucleotides that recruit endogenous human ADARs to edit endogenous transcripts in a simple and programmable way, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). We observed almost no off-target editing, and natural editing homeostasis was not perturbed. We successfully applied RESTORE to a panel of standard human cell lines and human primary cells and demonstrated repair of the clinically relevant PiZZ mutation, which causes α1-antitrypsin deficiency, and editing of phosphotyrosine 701 in STAT1, the activity switch of the signaling factor. RESTORE requires only the administration of an oligonucleotide, circumvents ectopic expression of proteins, and represents an attractive approach for drug development.

Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing-G/C Substitution Stabilizes GuideRNAs against Editing.

Site-directed RNA editing is an approach to reprogram genetic information at the RNA level. We recently introduced a novel guideRNA that allows for the recruitment of human ADAR2 to manipulate genetic information. Here, we show that the current guideRNA design is already able to recruit another human deaminase, ADAR1, in both isoforms, p110 and p150. However, further optimization seems necessary as the current design is less efficient for ADAR1 isoforms. Furthermore, we describe hotspots at which the guideRNA itself is edited and show a way to circumvent this auto-editing without losing editing efficiency at the target. Both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing.

Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy.

Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.

Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA.

Adenosine-to-inosine deamination can be re-addressed to user-defined mRNAs by applying phosphothioate/2'-methoxy-modified guideRNAs. Dense chemical modification of the guideRNA clearly improves performance of the covalent conjugates inside the living cell. Furthermore, careful positioning of a few modifications controls editing selectivity in vitro and was exploited for the challenging repair of the Factor 5 Leiden missense mutation.

Optimal guideRNAs for re-directing deaminase activity of hADAR1 and hADAR2 in trans.

Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5'-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5'-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5'-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together, our findings are very important to achieve selective and efficient editing in difficult codon and sequence contexts.