Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Elizabeth C Stahl; Jennifer K Sabo; Min Hyung Kang; Ryan Allen; Elizabeth Applegate; Shin Eui Kim; Yoonjin Kwon; Anmol Seth; Nicholas Lemus; Viviana Salinas-Rios; Katarzyna M Soczek; Marena Trinidad; Linda T Vo; Chris Jeans; Anna Wozniak; Timothy Morris; Athen Kimberlin; Thomas Foti; David F Savage; Jennifer A Doudna

doi:10.1016/j.ymthe.2023.06.019

Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Mol Ther. 2023 Aug 2;31(8):2422-2438. doi: 10.1016/j.ymthe.2023.06.019. Epub 2023 Jul 4.

Authors

Elizabeth C Stahl¹, Jennifer K Sabo², Min Hyung Kang³, Ryan Allen², Elizabeth Applegate², Shin Eui Kim², Yoonjin Kwon⁴, Anmol Seth⁴, Nicholas Lemus⁴, Viviana Salinas-Rios², Katarzyna M Soczek¹, Marena Trinidad², Linda T Vo², Chris Jeans⁵, Anna Wozniak⁶, Timothy Morris⁶, Athen Kimberlin⁶, Thomas Foti⁶, David F Savage⁷, Jennifer A Doudna⁸

Affiliations

¹ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA.
⁶ Aldevron LLC, Madison, WI 53719, USA.
⁷ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁸ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Gladstone Institutes, University of California, Berkeley, San Francisco, CA 94114, USA. Electronic address: doudna@berkeley.edu.

PMID: 37403358
PMCID: PMC10422012 (available on 2024-08-02)
DOI: 10.1016/j.ymthe.2023.06.019

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.

Keywords: CRISPR-Cas9; brain; endotoxin/LPS; genome editing; host immune response; microglia; mouse; neurons; non-viral delivery; viral vectors.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Animals
Brain / metabolism
CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
Gene Editing*
Mice
Ribonucleoproteins / metabolism

Substances

Ribonucleoproteins
CRISPR-Associated Protein 9

Grants and funding

F32 GM140637/GM/NIGMS NIH HHS/United States