We utilized a system for sequence-specific RNA base editing via Adenosine Deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). We systematically engineered it to harness ADARs, and comprehensively evaluated its specificity and activity in vitro and in vivo via two mouse models of human disease. We anticipate this platform will enable tunable and reversible engineering of RNAs for diverse applications.

Protein-based therapeutics can activate the adaptive immune system and lead to the production of neutralizing antibodies and to cytotoxic-T-cell-mediated clearance of the treated cells. Here, we show that the sequential use of immune-orthogonal orthologues of the CRISPR-associated protein 9 (Cas9) and of adeno-associated viruses (AAVs) eludes adaptive immune responses and enables effective gene editing from repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major-histocompatibility-complex proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors, and for 167 AAV VP1-capsidprotein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologs, which we validate for three Cas9 orthologs in mice, yet anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these ‘cloaked AAVs’ retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.

Targeted transcriptome engineering, in contrast to genome engineering, offers a complementary and potentially tunable and reversible strategy for cellular engineering. In this regard, adenosine to inosine (A-to-I) RNA base editing was recently engineered to make programmable base conversions on target RNAs. Similar to the DNA base editing technology, A-to-I RNA editing may offer an attractive alternative in a therapeutic setting, especially for the correction of point mutations. This Perspective introduces five currently characterized RNA editing systems and serves as a reader’s guide for implementing an appropriate RNA editing strategy for applications in research or therapeutics.

In the version of this Article originally published, in the heading for Fig. 1g 'DNA-targeting CRISPR effectors (285)' , '285' should have read '284'. In Fig. 3b,c,e and f, the final x axis label, ' AAV5-SaCas9' in blue, for the first dose was incorrect; the correct label is ' AAV5-SpCas9'. These figures have now been corrected. In addition, the caption for Supplementary Fig. 2 stated that 89 Cas13a, b and c orthologs were used; the correct number is 84. In Supplementary Table 1 the Cpf1 and Cas13 sequences were missing. The Supplementary Information and Supplementary Table files have now been corrected.