Testing AAV5 for activation of tdTomato in mouse airway

Experiment: Testing AAV5 for activation of tdTomato in mouse airway

PI: Guang-Ping Gao, PhD

Description: AAV2/5 mediated gene editing in the mouse airway was tested by deliverying SpCas9 and guide RNAs targeting the Ai9 transgene in Ai9 transgenic mice. Viral delivery was detected by GFP expression and gene editing quantified by tdTomato activation

Delivery Assays: Two AAVs encoding sgRNAs and SpCas9 were introduced by intratracheal injection into Ai9 mice. After 4 weeks, lung tissue was harvested and prepared by sectioning and immunohistochemical staining for GFP. Delivery efficency was calculated by counting GFP+ cells compared to DAPI stained cells.

Editing Assay: Two AAVs encoding sgRNAs and SpCas9 were introduced by intratracheal injection into Ai9 mice. After 4 weeks, lung tissue was harvested and prepared by sectioning and immunohistochemical staining for tdTomato. Editing efficiency calculated by counting fluorescent cells (RFP) compared to DAPI stained cells.

Parent Project: Develop Combinatorial Non-Viral and Viral CRISPR Delivery for Lung Diseases

Other experiments in this project: 2
- Testing AAV5 for activation of tdTomato in HEK293T cells
- Testing AAV5 for activation of tdTomato in mouse airway club and ciliated cells

Download: Submitted files

Validation:

Independent validation for Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9 mice

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Tissues

Epithelium of main bronchus

Cell Types

Epithelial cell of lung

Editors

SpCas9

Guide Target Locus

Ai9/Ai14 reporter transgene

Guides

sgAi9L

sgAi9R

Models

Ai9 mouse

Sex

Female

Vectors

AAV.pU1a-SpCas9

H509 AAVsc-u6-sgAI9L-U6-AI9R-U1A-EGFP (1)

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Results

Testing_AAV5_for_activation_of_tdTomato_in_mouse_airway_
												Editing Efficiency	Delivery Efficiency
Record Id	Condition	Tissue	Cell Type	Sex	Age	Editor	Model	Target Locus	Guide	Vector	Dosage	Injection Frequency	Editing Efficiency % cells	Delivery Efficiency % cells	Image
15000001632	Saline	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks		Ai9 mouse					once	0; 0; 0; 0; 0;
15000001633	Saline	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks		Ai9 mouse					once		0; 0; 0; 0; 0;
15000001634	AAV-SpCas9	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks	SpCas9	Ai9 mouse			AAV.pU1a-SpCas9	6e10 gc/each AAV per mouse by intratracheal injection	once	0; 0; 0; 0; 0;
15000001635	AAV-SpCas9	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks	SpCas9	Ai9 mouse			AAV.pU1a-SpCas9	6e10 gc/each AAV per mouse by intratracheal injection	once		0; 0; 0; 0; 0;
15000001636	Dual AAV	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks	SpCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	sgAi9L; sgAi9R	AAV.pU1a-SpCas9 ; H509 AAVsc-u6-sgAI9L-U6-AI9R-U1A-EGFP (1)	6e10 gc/each AAV per mouse by intratracheal injection	once	20.5; 21.5; 25.4; 23.1; 30.5;
15000001637	Dual AAV	epithelium of main bronchus (TARGET)	epithelial cell of lung	Female	8 weeks	SpCas9	Ai9 mouse	Ai9/Ai14 reporter transgene	sgAi9L; sgAi9R	AAV.pU1a-SpCas9 ; H509 AAVsc-u6-sgAI9L-U6-AI9R-U1A-EGFP (1)	6e10 gc/each AAV per mouse by intratracheal injection	once		30.2; 36.3; 29.1; 29.5; 33.2;

Dual AAV5 delivery of spCas9 and sgRNAs supports genome editing in mouse lung.

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Experiment: Testing AAV5 for activation of tdTomato in mouse airway

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Results