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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 Vivo
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[Validation]
Independent validation of Chen delivery platform using LNPs to deliver CRISPR/Cas9 to mouse inner ear
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Delivery of CRISPR/Cas9 via bioreducible lipid nanoparticles (LNPs) to the inner ear in Ai14 mice |
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2023-05-10
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In Vivo
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[Validation]
Independent validation of Chen delivery platform using LNPs to deliver CRISPR/Cas9 to mouse inner ear
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Delivery of CRISPR/Cas9 via bioreducible lipid nanoparticles (LNPs) to the inner ear in Ai14 mice. Subset of mice were administered LNP via canalostomy injection compared to uninjected control mice. Tissues were harvested 6 days after LNP administeration. On-target and off-target editing was assessed. |
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2023-07-11
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In Vivo
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[Validation]
Repeat experiment of independent validation of Chen delivery platform using LNPs to deliver CRISPR/Cas9 to mouse inner ear
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Repeat experiment of CRISPR/Cas9 delivery via bioreducible lipid nanoparticles (LNPs) to the inner ear in Ai14 mice. Subset of mice were administered LNP via canalostomy injection. Control mice were admininstered a blank LNP. Tissues were harvested 6 days after LNP administration. On-target editing was assessed by RFP (tdTomato) signal. |
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2023-07-11
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In Vivo
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[Validation]
Independent validation for McCray Delivery Team: Delivery of CRISPR Ribonucleoproteins to Airway Epithelia Using Novel Amphiphilic Peptides
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Ribonucleoproteins for CRISPR/Cas9 editing are complexed with amphiphilic peptides for delivery to lung airway epithilia via intranasal instillation into mTmG reporter mice. Editing is detected by production of GFP protein, and green fluorescence in airway linings |
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2021-09-07
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In Vivo
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[Validation]
Independent validation of Gong delivery platform using RNP-loaded nanocages to deliver CRISPR/Cas9 to mouse brain
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Delivery of CRISPR/Cas9 via RNP-loaded nanocages to the brain in Ai14 mice |
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2023-05-10
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In Vivo
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[Validation]
Independent validation of Gong delivery platform using RNP-loaded nanocages to deliver CRISPR/Cas9 to mouse brain
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Delivery of CRISPR/Cas9 via ribonuclear protein (RNP) loaded nanocages (NC) to the brain in Ai14 mice by intracranial bilateral injection. Tissues were harvested 14 days after NC administeration. On-target and off-target editing was assessed. |
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2023-07-18
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In Vivo
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[Validation]
Repeat experiment of independent validation of Chen delivery platform using LNPs to deliver CRISPR/Cas9 to mouse inner ear
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Delivery of CRISPR/Cas9 editor via bioreducible lipid nanoparticle to the inner ear in Ai14 mice |
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2023-05-10
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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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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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[Validation]
Independent validation of Sontheimer delivery platform using heavily modified guide RNAs complexed with Cas9 proteins to deliver CRISPR/Cas9 to mouse brain
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Heavily modified guide RNAs complexed with Cas9 proteins are injected locally to mouse striatum to activate reporter gene (mGFP). Editing detected via DAB staining in coronal brain sections. |
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2023-05-10
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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 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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[Validation]
Independent validation for Asokan Delivery Team: Evolving High Potency AAV Vectors for Neuromuscular Genome Editing.
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Quantification of CRISPR/Cas editing in liver and heart following custom AAV-mediated delivery. Detection of editing in non-target tissues. |
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2021-03-30
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In Vivo
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[Validation]
Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain
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Validation of delivery of AAV custom designed to cross the blood-brain barrier for CRISPR/Cas9 editing. Editing detected and quantified in brain by generation of tdTomato fluorescent protein signal from Ai9 reporter mice |
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2023-05-10
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In Vivo
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[Validation]
Independent validation for Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9 mice
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AAV5 encoding CRISPR/Cas editing machinery were delivered to the lungs of reporter mice by intratracheal instillation. After 4 weeks incubation, the mice were dissected and the lungs imaged for the presence of tdTomato fluorescence, indicating successful editing. Editing calculated by dividing the number of tdTomato+ red cells by the number of nuclei in each airway |
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2021-03-30
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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 Vivo
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[Validation]
Independent validation of Chaikof delivery platform using virus-like particle (VLP) delivery to the mouse liver
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Virus-like particles carrying a CRISPR/Cas base editor and a guide RNA targeting the PSCK9 locus were injected i.v. into male and female mice. One week after injection, the mice were dissected, and genomic DNA isolated from a panel of organs. Targeted NGS was performed to evaluate the degree of editing at the PCSK9 locus in the liver (primary target), and non-target organs. Two potential off-target editing sites (OT6 and OT7) were also sequenced. |
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2023-12-18
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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 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 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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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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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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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 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 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 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 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 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 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 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 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 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 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 Vitro
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Validating Gene Editing Reporter 14 (GER14) Mouse Model in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Gene Editing Reporter 14 (GER14) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with various CRISPR/Cas9 combinations (or Cre) and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for Katushka2S (RFP) fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced. ICE analysis tool from Synthego was used to score edits. NOTE: Some blasts did not PCR amplify well and it could not be determined if they had edits. Total blasts analyzed expressing the fluorescent reporter, 46-164. Total blast analyzed by Sanger sequencing, 39-142. |
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2024-02-23
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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 Vitro
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Testing newly discovered Biggie Phage editors in human cells
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2020-10-16
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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 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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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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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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