Single Nuclei RNA Sequencing of Mice Hippocampus to Evaluate in vivo Gene Editing by a Biodegradable Nanocage for CRISPR/Cas9 Ribonucleoproteins

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Date
2021Author
Araki, Alex Shintaro
Advisor(s)
Gimse, Kristan
Saha, Krishanu
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In recent years, CRISPR-Cas9 technology has shown promise as a versatile gene editing
tool that enables precise nucleotide base-editing (Sander & Joung, 2014), and are anticipated to
drive the next wave of gene therapies for a range of health conditions, including neurodegenerative
diseases. However, a major challenge in gene therapy development is in the generation of safe and
efficient non-viral delivery devices for in vivo Cas9-mediated gene editing. To date, very few safe
and effective non-viral Cas9 in vivo delivery methods have been reported, with most in vivo Cas9
delivery methods utilizing viral vectors that require extended manufacturing times with
questionable safety profiles and limited packing capacity (Miller et al., 2017). To address these
challenges, we have applied a previously developed nanocage (NC) design for non-viral in vivo
Cas9 ribonucleoprotein (RNP) delivery in mice brain. In vivo editing efficiency of RNPs delivered
with this NC (NC-RNPs) was evaluated in the hippocampi of Ai14 tdTomato reporter mice using
single nuclei RNA sequencing. Seurat integrated differential gene expression analyses between
NC-RNP Ai14 targeting experimental and NC-RNP scramble control hippocampi conditions
revealed a 2.8 fold increase in tdTomato expression in Ai14 targeting NC-RNP, indicating targeted
editing at the Ai14 locus. Upregulated tdTomato expression was observed in all cell types in the
hippocampal injection area, suggesting high penetrance of our NC-RNP formulation. Further
cluster annotation and gene expression analyses revealed no new immune cell activity between
experimental and control conditions, thereby supporting the safety of the NC for in vivo Cas9-RNP
delivery. While future RNAscope assays are necessary to confirm gene signatures we found
through snRNA-seq analyses, our data indicates a safe and robust non-viral Cas9-RNP delivery
method for in vivo brain editing.
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http://digital.library.wisc.edu/1793/84010Type
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