Delivery of saCas9 and gRNAs to nephron epithelia by AAV2 in kidney organoids.

Experiment: Delivery of saCas9 and gRNAs to nephron epithelia by AAV2 in kidney organoids.

PI: Ryuji Morizane, MD, PhD

Description: An AAV2 viral vector carrying SaCas9 and AAV2 vector expressing mCherry and carrying two gRNAs against the DMD gene were used in human kidney organoids to assess transgene delivery to nephron epithelia. Organoids were treated with both AAVs at MOI 10^5 for one week. Delivery of Cas9 and gRNA vectors to nephron cell types were evaluted by immunohistochemistry. Note: additional data not shown demonstrated mCherry and SaCas9 were not detectable in non-transduced cells.

Delivery Assays:

Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9+ mCherry+ fluorescent cells among CDH1+ stained loops of Henle/distal nephron cells
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9- mCherry+ fluorescent cells among PODXL+ stained podocytes
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9+ mCherry+ fluorescent cells among PODXL+ stained podocytes
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9+ mCherry+ fluorescent cells among LTL+ stained proximal tubule cells
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9+ mCherry- fluorescent cells among CDH1+ stained loops of Henle/distal nephron cells.
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9+ mCherry- fluorescent cells among PODXL+ stained podocytes
Calculated tranduction efficiceny of transgene delivery to nephron epithelia by counting Cas9- mCherry+ fluorescent cells among CDH1+ stained loops of Henle/distal nephron cells.

Parent Project: Vascularized kidney organoids on chip for efficacy and toxicity testing of somatic genome editing

Other experiments in this project: 6

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Editors

SaCas9 (AAV-SaCas9)

Guide Target Locus

DMD

Guides

DMD low gRNA-1

DMD low gRNA-2

Delivery Systems

AAV

Models

Human kidney organoid (stem cell-derived)

Vectors

AAV2-SaCas9

AAV2-gRNA

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Results

Delivery_of_saCas9_and_gRNAs_to_nephron_epithelia_by_AAV2_in_kidney_organoids.
								Delivery Efficiency
Record Id	Condition	Editor	Model	Delivery	Target Locus	Guide	Vector	Dosage	Delivery Efficiency % of LTL+ cells	Delivery Efficiency % of CDH+ cells	Delivery Efficiency % of PODXL+ cells	Image
15000003432	Cas9+, mCherry+ in LTL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5	33.3333333333333; 12.5; 54.5454545454545; 68.75; 57.1428571428571; 58.3333333333333; 29.4117647058824; 70; 60; 50; 25;
15000003433	Cas9+, mCherry+ in CDH1 Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5		10; 2.85714285714286; 4.54545454545455; 11.7647058823529; 2.85714285714286;
15000003434	Cas9+, mCherry+ in PODXL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5			1.61290322580645; 1.13636363636364; 0; 0; 0; 0; 0; 0; 0; 0; 0;
15000003435	Cas9+ mCherry- in LTL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5	0; 0; 4.54545454545455; 0; 0; 0; 0; 6.66666666666667; 4; 0; 8.33333333333333;
15000003436	Cas9+ mCherry- in CDH1 Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5		2.5; 0; 0; 5.88235294117647; 0;
15000003437	Cas9+ mCherry- in PODXL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5			0; 0; 0; 4.54545454545455; 0; 0; 0; 0; 0; 0; 0;
15000003438	Cas9- mCherry+ in LTL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5	0; 4.16666666666667; 4.54545454545455; 6.25; 9.52380952380952; 8.33333333333333; 5.88235294117647; 3.33333333333333; 2; 0; 0;
15000003439	Cas9- mCherry+ in CDH1 Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5		5; 2.85714285714286; 0; 0; 2.85714285714286;
15000003440	Cas9- mCherry+ in PODXL Stained Cells	SaCas9 (AAV-SaCas9)	Human kidney organoid (stem cell-derived)	AAV	DMD	DMD low gRNA-1; DMD low gRNA-2	AAV2-gRNA ; AAV2-SaCas9	MOI=10^5			0; 1.13636363636364; 0; 0; 0; 0; 0; 0; 0; 0; 0;

Evaluation on delivery of saCas9 and gRNAs by AAV2 in kidney organoids.

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Experiment: Delivery of saCas9 and gRNAs to nephron epithelia by AAV2 in kidney organoids.

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Results