Experiment: Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
PI: Aravind Asokan, PhD
Description: A dual vector strategy was employed: one delivering two single guide RNAs targeting the Rosa26 locus and one delivering CMV driven SaCas9 (both single stranded AAV cassettes). This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=5) by intravenous injection in Ai9 mice. A total dose of 4e12vg was injected into each mouse and vectors mixed in a 1:4 ratio of cas9 to guide RNA (1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector) and organs were harvested 4 weeks post injection. Editing efficency was determined by calculating percent TdTomato+ cells normalized to Dapi+ cells in liver and heart.
Editing Assay:
Editing efficiency calculated by counting fluorescent cells (TdTomato) compared to DAPI stained cells.
Parent Project: Evolving High Potency AAV Vectors for Neuromuscular Genome Editing
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Other experiments in this project: 6
- Testing virus region 8 (VR8) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
- Testing virus region 4 (VR4) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
- Cre Recombinase dose escalation study in Ai9 mice
- Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CB promoter)
- Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)
- Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CMV promoter) and self complementary sgRNA vector.
Download: Submitted files
Publications:
- Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. NCBI
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Results |
| Editing Efficiency | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Condition | Tissue | Cell Type | Sex | Editor | Model | Vector | Dosage | Injection Frequency | % of cells | Image |
| AAV9, Male | liver (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-Ai9-sgRNA1 + sgRNA2; AAV9-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAV9, Male | heart (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-Ai9-sgRNA1 + sgRNA2; AAV9-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAV9, Female | liver (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-Ai9-sgRNA1 + sgRNA2; AAV9-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAV9, Female | heart (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-Ai9-sgRNA1 + sgRNA2; AAV9-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAVcc47, Male | liver (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAVcc47-Ai9-sgRNA1 + sgRNA2; AAVcc47-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAVcc47, Male | heart (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAVcc47-Ai9-sgRNA1 + sgRNA2; AAVcc47-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAVcc47, Female | liver (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAVcc47-Ai9-sgRNA1 + sgRNA2; AAVcc47-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| AAVcc47, Female | heart (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAVcc47-Ai9-sgRNA1 + sgRNA2; AAVcc47-CMV-SaCas9 | 1e12vg of CMV Sacas9 vector and 3e12vg of the sgRNA vector | once | |||
| Mock-Neg Control | liver (TARGET) | Ai9 mouse | ||||||||
| Mock-Neg Control | heart (TARGET) | Ai9 mouse | ||||||||
Associated Publications |
| Publication Title |
|---|
| Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. NCBI |
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