In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

Mapping Cellular Reprogramming via Pooled Overexpression Screens with Paired Fitness and Single-Cell RNA-Sequencing Readout.

Understanding the effects of genetic perturbations on the cellular state has been challenging using traditional pooled screens, which typically rely on the delivery of a single perturbation per cell and unidimensional phenotypic readouts. Here, we use barcoded open reading frame overexpression libraries coupled with single-cell RNA sequencing to assay cell state and fitness, a technique we call SEUSS (scalable functional screening by sequencing). Using SEUSS, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs) and assayed the impact of TF overexpression on fitness and transcriptomic states. We further leveraged the versatility of the ORF library approach to assay mutant genes and whole gene families. From the transcriptomic responses, we built genetic co-regulatory networks to identify altered gene modules and found that KLF4 and SNAI2 drive opposing effects along the epithelial-mesenchymal transition axis. From the fitness responses, we identified ETV2 as a driver of reprogramming toward an endothelial-like state.

Oligonucleotide conjugated multi-functional adeno-associated viruses.

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.