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In Vitro
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Podocyte-specific gene editing in human kidney organoids
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Kidney organoids were derived from a human iPS cell line with Ai9 (tdTomato) fluorescence-on reporter knocked into the AAVS1 safe harbor locus. Intact kidney organoids were transfected with CRISPR ribonucleoprotein complexes with and without molecular targeting agent (MTA) specific for podocytes. Genome editing events were detected by induction of tdTomato from the Ai9 reporter. |
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2024-01-16
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In Vitro
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Imaging quantification of transfection efficiency with varying dosages of nanoparticles encapsulated with Cas9/sgRNA RNP on the liver-on-chip model system
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Liver-on-a-chip was used to examine the cellular uptake of CRISPR/Cas9 encapsulated nanoparticles. Two liver-targeted ligands were provided from the Gong Lab, RNP-NC attached to tri(GalNAc) and RNP-NC containing cell penetrating peptide (TAT). The triGalNAc is known to enhance RNP-NC target to hepatocytes, whereas TAT will enhance target and uptake of RNP-NC in all liver cells such as Kupffer cells. These ligands are tagged with Atto-550 a fluorescent protein reporter for easier detection. Liver microtissue with a monoculture of primary human hepatocytes (PHH) was used. Each well of the liver-on-a-chip typically is seeded with 6.0 × 10^5 hepatocytes 16 hours prior to the addition of RNP-NCs with flow of 1.0 µl/s through the liver 3D microtissues. Lipofectamine – RNP complex was prepared using Lipofectamine 2000 Transfection Reagent with 1:1 weight to weight ratio after optimization of transfection efficiency. The goal of the experiment was to examine transfection efficiency by testing two doses 2.4 ug and 24 ug RNP-NCs [tri(GalNAc), TAT] with the help of imaging. Transfection of 24µg RNP-NC shows higher uptake when compared to2.4µg RNP-NC. |
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2023-12-18
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In Vitro
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Quantification of transfection efficiency by flow cytometry with varying dosages of nanoparticles encapsulated with Cas9/sgRNA RNP on liver-on-chip model system
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Liver-on-a-chip was used to examine the cellular uptake of CRISPR/Cas9 encapsulated nanoparticles. Two liver-targeted ligands were provided from the Gong Lab, RNP-NC attached to tri(GalNAc) and RNP-NC containing cell penetrating peptide (TAT). The triGalNAc is known to enhance RNP-NC target to hepatocytes, whereas TAT will enhance target and uptake of RNP- NC in all liver cells such as Kupffer cells. These ligands are tagged with Atto-550 a fluorescent protein reporter for easier detection. Liver microtissue with a monoculture of primary human hepatocytes (PHH) was used. Each well of the liver-on-a-chip typically is seeded with 6.0 × 10^5 hepatocytes 16 hours prior to the addition of RNP-NCs with flow of 1.0 µl/s through the liver 3D microtissues. Lipofectamine – RNP complex was prepared using Lipofectamine 2000 Transfection Reagent with 1:1 weight to weight ratio after optimization of transfection efficiency. The goal of the experiment was to quantify transfection efficiency by testing two doses 2.4 ug and 24 ug RNP-NCs [tri(GalNAc), TAT] using flow cytometry. Transfection of 24µg RNP-NC shows higher uptake when compared to2.4µg RNP-NC. |
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2023-12-18
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In Vitro
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Testing different ratios of Lipofectamine-RNPs after 24 hours for determination of positive control
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Liver-on-a-chip was used to examine the cellular uptake of CRISPR/Cas9 encapsulated nanoparticles provided from the Gong Lab at the University of Wisconsin-Madison. The Gong lab conducted free radical polymerization of the monomer coating with (PEG)-acrylate to ensure that the RNP-NC be stable and able to conjugate different ligands. Two liver-targeted ligands were provided from the Gong Lab, RNP-NC attached to tri(GalNAc) and RNP-NC containing cell penetrating peptide (TAT). The tri(GalNAc) is known to enhance RNP-NC target to hepatocytes, whereas TAT will enhance target and uptake of RNP-NC in all liver cells such as Kupffer cells. These ligands are tagged with Atto-550 a fluorescent protein reporter for easier detection. The goal was to investigate cellular uptake of Cas9-gRNA nanocapsules using imaging after 24 hours and to determine the appropriate ratio for Lipofectamine-RNP positive control. |
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2023-12-18
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In Vitro
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Rhesus macaque CCR5 gRNA screening using ABE8e-Cas9
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Electroporation of plasmids containing ABE8e-Cas9 with 10 different gRNAs targeting CCR5 into rhesus primary skin fibroblasts. Editing efficiency was assessed using Next Generation Sequencing. |
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2023-03-15
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In Vivo
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Amphiphilic Peptides Deliver Base Editor RNPs to Rhesus Monkey Airway
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We utilized novel amphiphilic shuttle peptides to deliver base editor ribonucleoprotein (RNP) into the airways to edit airway epithelial cells (CCR5 locus) of rhesus monkeys. The Cas9-ABE8e RNP and shuttle peptides S10 or FSD315 were aerosolized into the rhesus monkey trachea. Seven days later, tissues were obtained and dissected, and airway epithelia collected from the trachea, mainstem, and segmental bronchi using cytology brushes. DNA was extracted from epithelial cells and subjected to high-throughput sequencing. Using the FSD315 shuttle peptide and Cas9-ABE8e, we achieved a mean editing efficiency of 2.8% at the CCR5 locus in airway epithelial cells (range 0.02 – 5.3%) depending on the anatomic region sampled. To visualize the biodistribution of the RNPs within the respiratory tract and in specific cell types, we delivered a Cy5-fluorescent peptide fused to a nuclear localization signal (NLS-Cy5) using the S10 peptide. The lungs were obtained 1 and 2 hours post-delivery, fixed, and examined by microscopy. Epifluorescence and confocal microscopy documented an effective intra-nuclear delivery of NLS-Cy5 into epithelial cells throughout the respiratory tract, including large, medium, and small airways, and alveolar regions. Ongoing analyses will identify the NLS-Cy5-positive epithelial cell types using co-localization with fluorescently-labeled antibodies. In summary, using a rhesus monkey model, following a single delivery of adenine base editor RNPs to the airways in a clinically relevant manner we achieved up to 5.3% editing efficiency of the CCR5 locus in airway epithelia, a level considered therapeutically relevant in cystic fibrosis. |
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2023-03-15
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In Vitro
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Delivery of Cas9 RNP using Shuttle peptide candidates to human airway epithelial cells cultured at the air liquid interface targeting CFTR locus
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Delivery of Cas9 RNP using Shuttle peptides candidates to human airway epithelial cells cultured at the air liquid interface targeting CFTR locus. Editing efficiency was assessed using NGS. |
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2023-03-15
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In Vitro
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Rhesus macaque CCR5 gRNA screening using ABE8e-Cas12a
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Electroporation of plasmids containing ABE8e-Cas12a with 11 different gRNAs targeting CCR5 into rhesus primary skin fibroblasts. Editing efficiency was assessed using high throughput DNA sequencing. |
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2023-03-15
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In Vitro
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Inhibitory effect of Cas9 RNP alone on S10- or FSDS315-mediated GFP protein delivery to HeLa cells
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Inhibitory effect of Cas9 RNP alone on S10- or FSD315-mediated GFP protein delivery to HeLa cells. GFP protein (10 μM), S10 or FSDS315 (10 μM) with or without Cas9 RNP (containing 2.5 μM Cas9 and 2 μM gRNA) were added to HeLa cells and GFP delivery quantified by flow cytometry. |
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2023-03-15
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In Vitro
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Delivery of ABE8e-Cas9 RNP using Shuttle peptide candidates to human airway epithelial cells cultured at the air liquid interface targeting B2M locus
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Delivery of ABE8e-Cas9 RNP using Shuttle peptides candidates to human airway epithelial cells cultured at the air liquid interface targeting B2M locus. Editing efficiency was assessed using Sanger sequencing. |
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2023-03-15
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In Vitro
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Delivery of ABE8e-Cas9 RNP using Shuttle peptide candidates to rhesus monkey airway epithelial cells cultured at the air liquid interface targeting CCR5 locus.
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Delivery of ABE8e-Cas9 RNP using Shuttle peptides candidates to rhesus monkey airway epithelial cells cultured at the air liquid interface targeting CCR5 locus. Editing efficiency was assessed using Next Generation Sequencing. |
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2023-03-15
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In Vivo
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On-target editing compared to 14 circle-seq nominated off-target sites of adenine base editor delivered by BE-eVLP vs AAV in the C57BL/6 liver
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On-target editing compared to off-target editing at 14 CIRCLE seq nominated sites in livers of an adenine base editor delivered by engineered virus-like particles (BE-eVLPs). Treated mice vs. untreated vs. AAV was assessed one week after systemic administration of BE-eVLPs or AAV-Pcsk9 to C57BL/6 mice. DNA sequencing reads containing A-T to G-C mutations within protospacer positions 4-10. |
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2022-04-15
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In Vivo
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FUS (focused ultrasound) array validation in Ai9 mice
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9.3 week-old Ai9 mice (4 male and 4 female) were administered Ai9-targeting SaCas9 AAV9 vector through intravenous adminsitration (2E12 vg/mouse) and left hemisphere was targeted by FUS (focused ultrasound) array for BBB (blood brain barrier) opening |
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2022-04-15
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In Vivo
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Pcsk9 adenine base editor efficiency in liver and nonliver tissue
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Adenine base editing at the Pcsk9 exon 1 splice donor site in mouse heart, kidney, liver, lungs, muscle, and spleen was assessed one week after systemic administration of an adenine base editor delivered by engineered virus-like particles (BE-eVLPs) in C56BL/6 mice |
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2022-04-15
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In Vivo
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Testing preparation for independent validation at The Jackson Laboratory Small Animal Testing Center
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Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain |
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2021-10-01
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In Vivo
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Testing AAV5 for activation of tdTomato in mouse airway club and ciliated cells
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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. Gene editing quantified by tdTomato activation and cell specific markers for club and ciliated cell types. |
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2021-09-21
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In Vivo
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Cre Recombinase dose escalation study in Ai9 mice
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A single stranded cmv cre cassette was packaged into AAV9 or AAVcc47 and injected intravenously in Ai9 mice. We injected n=3 at three different doses (1e10, 1e11, 1e12 vg) and harvested organs 4 weeks post injection. Fluorescence intensity in liver, heart, and skeletal muscle was quantified with tiff based images in Image J and neuronal transduction from each vector was quantified at the 1e12vg dose by counting the number of tdTomato+ neurons and number of NeuN+ cells from multiple sections and images. |
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2021-09-16
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
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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. |
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2021-09-16
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)
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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=3) and AAVcc47 (n=3) by intravenous injection in Ai9 mice. A total dose of 3e12vg was injected into each mouse (1.5e12vg each vector mixed 1:1) 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. |
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2021-09-16
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In Vivo
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Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CB promoter)
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A dual vector strategy was employed: one delivering a single guide RNA and CB driven SaCas9, and another delivering the second guide RNA and CB driven SaCas9. This strategy was evaluted with both AAV9 (n=4) and AAVcc47 (n=5) by intravenous injection in Ai9 mice. A total dose of 2e12vg was injected into each mouse (1e12vg each vector mixed 1:1) 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. |
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2021-09-16
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In Vivo
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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.
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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. |
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2021-09-16
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In Vivo
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Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.
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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. |
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2021-04-17
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In Vitro
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Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro
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Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts |
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2021-04-17
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In Vitro
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Testing newly chemically modified crRNA and tracrRNA in mTmG mouse embryonic fibroblasts
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Chemically modified crRNA and tracrRNA were delivered by electroporation to embryonic fibroblasts harvested from the mTmG reporter mouse. Gene editing was determined by reporter activation. |
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2021-04-15
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In Vivo
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Delivery of unmodified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA using the S10 shuttle peptide to activate the mTmG reporter in mouse brain
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Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged. |
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2021-04-15
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In Vivo
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Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
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Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged. |
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2021-04-15
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In Vitro
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Testing newly chemically modified crRNA and tracrRNA to activate the TLR1 reporter in human cells
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Chemically modified crRNA and tracrRNA were delivered by electroporation to transgenic human HEK-293T cells harboring the TLR1 reporter. Gene editing was determined by reporter activation. |
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2021-04-15
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In Vitro
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Testing newly chemically modified crRNA and tracrRNA to activate the TLR reporter in human cells
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Chemically modified crRNA and tracrRNA were delivered by electroporation in presence of amphiphilic peptide to transgenic human HEK-293T cells harboring the TLR-MCV1 reporter. Gene editing was determined by reporter activation. |
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2021-04-15
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In Vitro
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Testing newly chemically modified crRNA and tracrRNA in mouse Neuro 2A cells
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Chemically modified crRNA and tracrRNA were delivered by electroporation to mouse Neuro2A cells. Editing activity was determined by Sanger sequencing |
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2021-04-15
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In Vitro
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Testing newly chemically modified crRNA and tracrRNA in mouse Hepa 1-6 cells
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Chemically modified crRNA and tracrRNA were delivered by electroporation to mouse Hepa 1-6 cells. Editing activity was determined by Sanger sequencing |
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2021-04-15
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In Vivo
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Delivery of unmodified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
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Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged. |
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2021-04-15
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In Vivo
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Delivery of RNP containing chemically modified crRNA C20 with chemically modified tracrRNA T2-PS to determine the RNP distribution in TLR-MCV mouse brain
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Chemically modified crRNA and tracrRNA were injected into the striatum of TLR-MCV mice and the distribution of RNP was imaged. |
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2021-04-15
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In Vivo
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Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
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Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged. |
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2021-04-15
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In Vivo
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Testing new LNPs (lipid nanoparticles) for delivery of Cas9 mRNA/sgRNA in adult mouse cochlea
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Delivery of Cas9/sgRNA mRNA via new LNPs to the cochlea by cochleostomy and gene editing is measured by percentage of tdTomato positive cells. |
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2021-04-13
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In Vitro
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Testing new LNPs (lipid nanoparticles) for delivery of Cas9 mRNA/sgRNA in primary fibroblast cells from adult Ai14 mouse cochlea
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LNP delivery of Cas9 mRNA/sgRNA in primary fibroblast cells from adult Ai14 mouse cochlea (inner ear) |
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2021-04-13
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In Vivo
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Testing new LNPs (lipid nanoparticles) for delivery of Fluc mRNA in adult mice
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Delivery of firefly luciferase mRNA via new Lipid NanoParticles by tail vein injection into WT C57BL/6J mice targeting the Liver and delivery is measured by luciferase expression. |
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2021-04-13
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In Vivo
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Testing virus region 8 (VR8) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
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C57BL/6 mice (N=3) were injected intravenously at a dose of 5e13 vg/kg per mouse with a self-complementary AAV9 or ccAAV vector encoding a GFP reporter. The biodistribution of of virus transduction was chacterized in various tissues and cell types by fluorescence imaging quantification. |
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2020-11-19
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In Vivo
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Testing virus region 4 (VR4) mutant cross-species compatible Adeno Associated Viruses (ccAAVs) in mice.
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C57BL/6 mice (N=3) were injected intravenously at a dose of 5e13 vg/kg per mouse with a self-complementary AAV9 or ccAAV vector encoding an mCherry reporter. The biodistribution of of virus transduction was chacterized in various tissues and cell types by fluorescence imaging quantification. |
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2020-11-19
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In Vitro
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Gene editing in vitro by various peptide variants delivering Cas RNPs to primary Human airway epithelia cells.
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In Vitro shuttle peptide delivery of Cas12a RNPs targeting human CFTR and HPRT genes in human primary airway epithelia. Editing efficiency was assessed after 72hrs by sanger sequencing. |
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2020-11-02
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In Vitro
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Peptide Shuttle optimization to deliver Cas9 RNP to human airway epithelia cells
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Delivery of Cas9 RNP targeting human CFTR in Primary human epithelia cells. Gene editing efficiency was determined by percentage of NGS reads that showed an indel |
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2020-11-02
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In Vivo
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Testing Shuttle Peptides ability to deliver GFP-NLS to airway epithelia.
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Delivery of GFP via shuttle peptides to mouse airway epithelium via nasal instilation. Delivery efficiency was quantified in large and small airways by counting the number of GFP positive cells divided by the number of DAPI cells. |
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2020-11-02
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In Vivo
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Shuttle peptides enable in vivo gene editing with Cas9 and Cas12a RNP in mouse airway epithelia
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In vivo shuttle peptide delivery of Cas9 and Cas12a RNPs in mouse airway epithelia. Gene editing was quantified by the GFP+ cells in large and small airways following 1 delivery of GFP protein by GFP positive cells compared to DAPI stained cells. |
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2020-11-02
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In Vitro
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Peptide Shuttle optimization to deliver Cas12a RNP to human airway epithelia cells
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Delivery of Cas12a RNP targeting human CFTR in Primary human epithelia cells. Gene editing efficiency was determined by percentage of NGS reads that showed an indel |
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2020-11-02
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In Vitro
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Gene editing in vitro by various peptide variants delivering Cas12a in NK cells.
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In Vitro shuttle peptide delivery of Cas12a RNP targeting human NKG2A gene in human NK cells. Gene editing was assessed after 48hr by T7E1 digestion. |
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2020-11-02
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In Vivo
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Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain.
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Nanocapusules carrying CRISPR Cas9 RNP with guide RNA targeting the stop sequence in the Ai14 transgene are intracerebrally delivered to Ai14 mice and gene editing is measured by gain of tdTomato protein expression. |
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2020-10-28
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In Vitro
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Testing AAV5 for activation of tdTomato in HEK293T cells
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AAV shuttle plasmids expressing SpCas9 and guide RNAs targeting the Ai9 transgene were tested in HEK293T cells by transient transfection. Both delivery and gene editing were detected by fluorescence. |
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2020-10-20
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In Vivo
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Testing AAV5 for activation of tdTomato in mouse airway
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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 |
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2020-10-20
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