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Experiment: Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.

PI:   Benjamin E Deverman, PhD>

Description: 3e11 vg/mouse of AAV-BI28:GFAP-SaCas9-WPRE-pA and 3e11 vg/mouse of AAV-BI28:GFAP-NLS-GFP-U6-L1-U6-R2 were codelivered intravenously to adult male and female Ai9 mice. Editing was assessed in brain sections 4 weeks later.
Editing Assay: Editing efficiency assessed by quantifying tdTomato positive area above threshold
Download: Submitted files
Filters ..
  Tissues
Nervous
  • Cerebral cortex (astrocyte)  (TARGET)
  • Dorsal plus ventral thalamus (astrocyte)  (TARGET)
  • Striatum (astrocyte)  (TARGET)
Cell Types
 astrocyte
Editors
 SaCas9
Guide Target Locus
 Ai9/Ai14 reporter transgene
  Guides
 L1-modified
 R2-modified
Models
 Ai9 mouse
  Sex
 Female
 Male
  Vectors
 BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2
 BI28:AAV-GFAP-SaCas9-WPRE3-pA
Select experimental variable to highlight records on the chart: 
 

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Results
Testing_gRNA_sequence_and_gRNA_scaffold_modified_in_Ai9_mice.
Editing Efficiency
Record Id Condition Tissue Cell Type Sex Editor Model Target Locus Guide Vector Dosage Injection Frequency Editing Efficiency tdTomato positive area Image
15000000949 Male cerebral cortex (TARGET) astrocyte Male SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once
15000000950 Male dorsal plus ventral thalamus (TARGET) astrocyte Male SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once
15000000951 Male striatum (TARGET) astrocyte Male SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once
15000000952 Female cerebral cortex (TARGET) astrocyte Female SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once
15000000953 Female dorsal plus ventral thalamus (TARGET) astrocyte Female SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once
15000000954 Female striatum (TARGET) astrocyte Female SaCas9 Ai9 mouse Ai9/Ai14 reporter transgene L1-modified; R2-modified BI28:AAV-GFAP-NLS-GFP-WPRE-synpA-L1-R2; BI28:AAV-GFAP-SaCas9-WPRE3-pA 3e11 vg each virus once

Experiment Wide Protocols

Title Description File Download SCGE ID
Deverman_Comprehensive Methods Procedure for plasmid cloning, editing evaluation in fibroblast, AAV production and administration, tissue processing and IHC. Deverman_Comprehensive_methods.pdf 21000000012
Deverman_Area Based Quantification of Editing Efficiency Protocol Procedure for non-IHC based image quantification of editing. Deverman_Area_based_quantification_of_editing_efficiency.pdf 21000000011
In vivo editing with the AAV-BI28 capsid and third generation GFAP-SaCas9 vectors

In vivo editing with the AAV-BI28 capsid and third generation GFAP-SaCas9 vectors. Ai9 mice were injected with BI28:GFAP-SaCas9 (3E11vg/mouse) and BI28:GFAP-NLS-GFP-U6-gRNA(L1- R2) (3E11 vg/mouse) and expression and editing were assessed 4 weeks later. A. Whole brain section and Ai9 locus editing (tdTomato) enabled by AAV-BI28:GFAP-Cas9. B. Efficient co-transduction of BI28:GFAP-SaCas9 and BI28:GFAP-NLS-GFP-gRNA-L1-R2 in cortical astrocytes (S100). C. The graph shows quantification of CNS astrocyte editing measured by the fraction of tdTomato+ area above threshold within the indicated brain regions (mean ± SD, n=6 females and n=4 males). D. Image shows transduction and editing in the liver.