Experiment: 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.
PI: Aravind Asokan, PhD
Description: A dual vector strategy was employed: one self complementary vector delivering two single guide RNAs targeting the Rosa26 locus and one delivering CMV driven SaCas9 (single stranded vector). This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=4) by intravenous injection in Ai9 mice. A total dose of 4e12vg was injected into each mouse and vectors mixed in a 1:1 ratio of cas9 to guide RNA (2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary 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
- 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 efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
- 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.
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 | Sex | Editor | Model | Vector | Dosage | Injection Frequency | % of cells | Image |
| AAV9, Male | liver (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAV9_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAV9, Male | heart (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAV9_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAV9, Female | liver (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAV9_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAV9, Female | heart (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAV9_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAVcc47, Male | liver (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAVcc47_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAVcc47, Male | heart (TARGET) | Male | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAVcc47_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAVcc47, Female | liver (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAVcc47_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| AAVcc47, Female | heart (TARGET) | Female | SaCas9-Lagor | Ai9 mouse | AAV9-CMV-SaCas9; AAVcc47_pTR_self comp 2xU6-Ai9 guides | 2e12vg of CMV Sacas9 vector and 2e12vg of the self complementary sgRNA vector | once | ||
| Mock | 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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