Targeted high-fidelity integration of DNA elements into the genome - SUMMARY Genome editors can be used to permanently disrupt protein-coding genes via indels, change specific bases, and, with the advent of Prime editing, make small deletions, substitutions, and insertions. However, none of these existing technologies can efficiently and precisely introduce DNA templates with modified bases into the genome of somatic cells. Targeted installation of DNA templates with modified bases could enable new ways to interrogate genome function. And installation of regulatory elements, such as methylated CpG islands, enhancers, promoters, and polyadenylation (polyA) transcription termination signals, could be used to control gene expression. In preliminary studies, we found that zinc finger nucleases (ZFNs) can be used to simultaneously carry a DNA template into the nucleus and facilitate high-fidelity directional integration of the DNA template at the sticky (cohesive) ends generated by the ZFN pair. The main components of this “sticky trap” (ST) genome engineering technology include a genome editor that generates sticky ends and double-stranded ST- oligonucleotides (ST-oligos). ST-oligos are comprised of nuclease-protected sticky ends, a DNA payload and a sequence motif that promotes high-efficiency targeted integration into dividing and terminally differentiated cells. Our research proposal is focused on testing the hypothesis that ST technology can be harnessed to disrupt Ube3a-ATS, a long non-coding RNA (lncRNA) associated with Angelman syndrome (AS). Permanent disruption of Ube3a-ATS has the potential to enduringly treat this debilitating neurogenetic disorder that is caused by loss of maternal Ube3a in neurons. In this proposal, we will complete the following aims: 1) Install a ST-oligo polyA element to prematurely terminate the therapeutically relevant lncRNA (Ube3a-ATS) in cultured neurons. We and others previously showed that premature termination of Ube3a-ATS can unsilence the functional paternal Ube3a allele in AS model mice. In preliminary studies, we generated small synthetic polyA sequences that efficiently disrupts gene expression as well as ZFN pairs that can be used to directionally integrate ST-oligos into mouse Ube3a-ATS. 2) Adapt ST-oligos for use with CRISPR/Cas genome editors that generate sticky ends. This aim will broaden the utility of ST-oligo technology beyond ZFNs. 3) Evaluate the extent to which ST components disrupt Ube3a-ATS and unsilence paternal Ube3a in vivo. We will deliver sticky trap components (ZFN or Cas mRNAs, ST-oligo) in lipid nanoparticle and proteolipid vehicle formulations to limit the amount of time the genome editor remains active in the brain. The ability to target DNA integration precisely and efficiently in dividing and non-dividing cells, combined with the relative ease of manufacturing lipid-based particles and nucleic acids makes our approach broadly useful for addressing basic research questions and for treating genetic disorders and diseases.
