Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Stahl, E. C.; Sabo, J. K.; Kang, M. H.; Allen, R.; Applegate, E.; Kim, S. E.; Kwon, M.; Seth, A.; Lemus, N.; Salinas-Rios, V.; Soczek, K.; Trinidad, M.; Vo, L. T.; Jeans, C.; Wozniak, A.; Morris, T.; Kimberlin, A.; Foti, T.; Savage, D. F.; Doudna, J. A.

doi:10.1101/2023.04.13.536294

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bioRxiv · 10.1101/2023.04.13.536294

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

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Abstract

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here we tested the ability of cell penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low-endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

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Stahl, E. C., Sabo, J. K., Kang, M. H., Allen, R., Applegate, E., Kim, S. E., Kwon, M., Seth, A., Lemus, N., Salinas-Rios, V., Soczek, K., Trinidad, M., Vo, L. T., Jeans, C., Wozniak, A., Morris, T., Kimberlin, A., Foti, T., Savage, D. F., Doudna, J. A.. 2023-04-13. Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs. https://doi.org/10.1101/2023.04.13.536294

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