Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes
Open accessAbstract
We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.
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Direct application of pre-assembled CRISPR-Cas RNPs can reduce possible off-target mutations relative to delivering Cas9-encoding DNA or mRNA due to the short half-life of RNPs.
“This delivery mode can reduce possible off-target mutations relative to delivering Cas9-encoding DNA or mRNA due to the short half-life of RNPs 76,85-87 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [76]
The Abstract (page unavailable) says that off-target editing is a concern with genetically encoded Cas9 delivered by viral vectors or plasmids, and that short-lived, preassembled Cas9 RNPs overcome this concern. It does not make the same comparison with Cas9-encoding mRNA, so that part of the claim is unsupported. (LLM, conf=0.85)
Experimental — accuracy not yet measuredThe total number of cells counted from every 4th DAPI stained brain slice was multiplied by 12 to obtain the estimate of the total number of tdTomato+; DAPI+ cells in the striatum or hippocampus, as similar to previously reported methods.
“Then, the total number of cells counted from every 4th DAPI stained brain slice was multiplied by 12 to obtain the estimate of the total number of tdTomato+; DAPI+ cells in the striatum or hippocampus, as similar to previously reported methods (37).”
From Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection (2023) · cites this paper as [37]
The Additional information cell-counting passage describes staining sampled brain sections with DAPI, counting tdTomato/DAPI double-positive cells, and scaling the count by section periodicity. That supports the claimed similarity of method, but this paper sampled every sixth dorsal-striatum section and multiplied by 6—not every fourth section and multiplied by 12. The cited passage does not describe this counting procedure for the hippocampus. No page number is provided in our copy. (LLM, conf=0.90)
Experimental — accuracy not yet measuredDisadvantages of LNP delivery include significant toxicity and sometimes undesired selectivity of cell-type specific uptake.
“Disadvantages of this approach include significant toxicity of the LNPs 76 and the sometimes undesired selectivity of cell-type specific uptake of the particles.”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [76]
Direct injection of 4xNLS-Cas9-2xNLS RNPs into the striatum and hippocampus of Ai14 mice edited genes in brain tissue efficiently.
“Specifically, Staahl et al. (2017) demonstrated that direct injection of the Cas9 protein fused to 6 SV40 nuclear localization signals (4xNLS-Cas9–2xNLS), complexed to sgRNA (4xNLS-Cas9–2xNLS RNPs) into the striatum and hippocampus of Ai14 mice was able to edit genes in brain tissue efficiently (37).”
From Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection (2023) · cites this paper as [37]
MC3 LNP Cas9 mRNA/Ai9 sgRNA demonstrates similar or better local transfection efficiency in the striatum and the hippocampus compared to previously demonstrated results using Cas9 RNPs (37, 43).
“MC3 LNP Cas9 mRNA/Ai9 sgRNA demonstrates similar or better local transfection efficiency in the striatum and the hippocampus compared to previously demonstrated results using Cas9 RNPs (37, 43).”
From Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection (2023) · cites this paper as [37]
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References 30 · Cited by 355
- Authors
- Staahl, B. T., Benekareddy, M., Coulon-Bainier, C., Banfal, A. A., Floor, S. N., Sabo, J. K., Urnes, C., Munares, G. A., Ghosh, A., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- F. Hoffmann-La Roche AG (CH)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A
- Venue
- Nature Biotechnology
- Published
- 2017-02-13
- Volume
- 35
- Issue
- 5
- Pages
- 431-434
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6649674
- OpenAlex
- W2588309240
- Cited by
- 355
- References
- 30
- Citations by year
- 2026: 23 · 2025: 36 · 2024: 29 · 2023: 40 · 2022: 35 · 2021: 29 · 2020: 45 · 2019: 39 · 2018: 58 · 2017: 19 · 2013: 1 · 2012: 1
- FWCI
- 17.97
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- F. Hoffmann-La Roche
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- genome editing, Cas9 ribonucleoprotein, nuclear localization signal, postmitotic neurons, adult mouse brain, hippocampus, striatum, cerebral cortex, in vivo gene delivery, neurological disease therapy
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Animals, Bacterial Proteins, Brain, Endonucleases, Male, Nerve Tissue Proteins, Ribonucleoproteins, Protein Engineering, Gene Targeting, Mice, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Proteins
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