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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1653: Transcriptomic Profiling of the Mouse Hippocampus After Intracerebral Injection of Cas9 Nanocapsule Genome Editors

Type: Poster Session

Poster Board Number: 1653
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Evaluating the editing efficiency and cell-type specificity of genome editors is a critical task for developing somatic cell genome editing strategies, especially for those that target the brain. Standard methods rely on deep sequencing at the on-target site combined with immunohistochemistry within treated animal model systems to enumerate the types of edited cells in select tissues after administering genome editors. Animal reporter systems that express fluorescent proteins after successful on-target genomic editing provide robust platforms to evaluate the number of edited cells but typically incorporate limited opportunities to co-register cell-type markers with the reporter protein. Thus, molecular characterization of an edited cell typically is limited to 2-4 cell-type markers that can be imaged simultaneously with the fluorescent reporter. Here, we report on a new approach to deeply characterize the transcriptome of edited cells within the brain of Ai14-tdTomato reporter mice. We performed single nucleus RNA sequencing (snRNA-seq) on nuclei isolated from hippocampi after intracranial injection of nanoparticle genome editors into the dorsal hippocampus. We utilized a previously developed biodegradable nanocage (NC) capable of delivering a preassembled SpyCas9 protein-gRNA ribonucleoprotein complex (RNP). RNPs targeting the Ai14 loxP-STOP cassette were encapsulated into these NCs and delivered into the dorsal hippocampus via intracranial injection. After performing snRNA-seq, we observe the capture of ~500-15000 unique transcripts per nuclei and robust Ai14-tdTomato reporter expression in nuclei from neuronal, glial, and oligodendrocytic cells. Additionally, differential cell-type specific transcriptomic shifts were identified primarily in immune and cell signaling pathways between treatment with recombinant adeno-associated viral vectors (rAAV) or NC. Overall, transcriptional profiling provides a high-resolution and complementary method to examine the cellular outcomes from genome editing within animal reporter systems, which has high potential to inform the clinical development of genome editing therapeutics.

Kirstan Gimse1, Yuyuan Wang1, Jesi Felton2, Jeanette Metzger2, Marina Emborg2, Jon Levine2, Shaoqin Sarah Gong1, Krishanu Saha1

1Wisconsin Institute for Discovery, University of Wisconsin- Madison, Madison, WI,2Wisconsin National Primate Research Center, University of Wisconsin- Madison, Madison, WI
 K. Gimse: None.

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