43 results for Ai9

Ai9 mouse

Model System  - [In Vivo] [Small Animal Testing Center (SATC), AAV tropism, Delivery Systems Initiative] [Mouse]
Matched on: name -->  Ai9 mouse  -->  Ai9 mouse
Ai9 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus.
Show Experiments (10)

Testing gRNA sequence and gRNA scaffold modified 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)
Testing AAV5 for activation of tdTomato in mouse airway
Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)
AAV Tropism project
Cre Recombinase dose escalation study in Ai9 mice
Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain
Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
Testing AAV5 for activation of tdTomato in mouse airway club and ciliated cells
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.

Ai9 SaCas9 Guide A

Guide  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on: name -->  Ai9 SaCas9 Guide A  -->  Ai9 SaCas9 Guide A  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B
This gRNA targets the Ai9 and related transgenes

Ai9 mouse (BCM)

Model System  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on: name -->  Ai9 mouse (BCM)  -->  Ai9 mouse (BCM)  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B
Ai9 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus.

Ai9-SauSpyCas9 mouse

Model System  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on: name -->  Ai9-SauSpyCas9 mouse  -->  Ai9-SauSpyCas9 mouse
Ai9-SauSpyCas9 is a version of Ai9 that has a single guide RNA sites on both sides of the STOP cassette. This is a Cre reporter allele that has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. Ai9 mice express robust tdTomato fluorescence following Cre-mediated recombination.

Ai9 SaCas9 Guide B

Guide  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on: name -->  Ai9 SaCas9 Guide B  -->  Ai9 SaCas9 Guide B  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B
This gRNA targets the Ai9 and related transgenes

Ai9 mouse immortalized fibroblasts

Model System  - [In Vitro] [Delivery Systems Initiative] [Mouse]
Matched on: name -->  Ai9 mouse immortalized fibroblasts  -->  Ai9 mouse immortalized fibroblasts
Immortalized fibroblasts made from Ai9 (B6.Cg-Gt(ROSA)26Sor^tm9(CAG-tdTomato)Hze/J) mice

HEK-293T with Ai9 transient reporter assay

Model System  - [In Vitro] [Delivery Systems Initiative] [Human]
Matched on:  -->  HEK-293T with Ai9 transient reporter assay
HEK-293T cells transfected with an Ai9 inducible transgene reporter plasmid used to test gene editing activity by fluorescence. HEK293T is an epithelial-like cell that was isolated from the kidney of a patient.

Cre Recombinase dose escalation study in Ai9 mice

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Cre Recombinase dose escalation study in Ai9 mice
Asokan Aravind  Last Updated Date: 2021-09-16
 
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.

Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Testing gRNA sequence and gRNA scaffold modified in Ai9 mice.
Deverman Benjamin E  Last Updated Date: 2021-04-17
 
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.

Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro

Experiment  - [In Vitro] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus
Deverman Benjamin E  Last Updated Date: 2021-04-17
 
Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts

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

Experiment  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9
Heaney Jason D  Last Updated Date: 2021-03-30
 
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

Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9
Asokan Aravind  Last Updated Date: 2021-09-16
 
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.

Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to
Asokan Aravind  Last Updated Date: 2021-09-16
 
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.

Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CB promoter)

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to
Asokan Aravind  Last Updated Date: 2021-09-16
 
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.

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.

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to
Asokan Aravind  Last Updated Date: 2021-09-16
 
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.

[Validation] Independent validation of Gong delivery platform using RNP-loaded nanocages to deliver CRISPR/Cas9 to mouse brain

Experiment  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Murray Stephen A  Last Updated Date: 2023-05-10
 
Delivery of CRISPR/Cas9 via RNP-loaded nanocages to the brain in Ai14 mice

Murray-SATC_Gong-Validaiton_Gong Study Protocol

Protocol  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Procedure for intracranial injection, immunofluorescence and imaging.

sNLS-SpCas9-sNLS

Genome Editor  - [In Vivo] [Small Animal Testing Center (SATC), Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
SpCas9 with N- and C-terminal SV40 NLS

RNP-NC-RVG

Delivery System  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. This nanoparticle has an addition of a RVG peptide YTIWMPENPRPGTPCDIFTNSRGKRASNG which specifically interacts withthe N-acetylecholine receptor (AchR) on neuronal cells, which mediates NP entry

AB_2209751 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: Rockland Cat# 600-401-379 
Anti-RFP (RABBIT) Antibody

RRID:AB_2813835 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain.

Experiment  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
Gong Shaoqin (Sarah)  Last Updated Date: 2020-10-28
 
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.

AB_2532994 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: 13-0300  
Thermo-Fisher, Rat anti-GFAP

SauCas9

Genome Editor  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B

Ai14 gRNA

Guide  - [In Vivo] [Small Animal Testing Center (SATC), Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
This sgRNA targets the Ai9 and related transgenes at multiple sites

Gong_Intracranial Injection Procedure for Mice

Protocol  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
Procedure for intracranial delivery to mouse brain.

Murray-SATC_Gong-Validation Brain Injection in Mice Protocol

Protocol  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Procedure for brain injection surgical procedure, pre- and post-operative care for mice.

AB_10711040 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: ab104224  
Abcam, mouse anti-NeuN

AB_141637 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: A21207 
Invitrogen, Donkey anti-rabbit alexa fluor 594

AB_141607 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: A21202 
Invitrogen, Donkey anti-mouse alexafluor 488

RRID:AB_2209751 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

AAVcc47-SaCas9-Ai9

Vector  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B
AAV2/9 expressing SaCas9 and single sgRNA under U6 promoter

Ai14 mouse (congenic)

Model System  - [In Vivo] [Small Animal Testing Center (SATC), Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
Ai14 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. The att site flanked neo selection cassette has been removed in this strain.

RNP-NC-CPP

Delivery System  - [In Vivo] [Small Animal Testing Center (SATC), Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. This nanoparticle has an addition of a cell penetrating peptide (CPP) from the TAT peptide (GRKKRRQRRRPQ) which lacks cell-type specficity

RNP-NC-no ligand

Delivery System  - [In Vivo] [Small Animal Testing Center (SATC), Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA.

Ai14 mouse

Model System  - [In Vivo] [Delivery Systems Initiative] [Mouse]
Matched on:  -->  Ai14 gRNA
Ai14 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. The att site flanked neo selection cassette has been removed in this strain.

AB_2813835 

Antibody  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id: ab150155 
Abcam, Donkey anti-rat alexa fluour 647

RRID:AB_2532994 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

RRID:AB_10711040 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

RRID:AB_141637 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

RRID:AB_141607 

Antibody  [Mouse]
Matched on:  -->  Ai14 gRNA
Other Id:  

[Validation] Independent validation for Asokan Delivery Team: Evolving High Potency AAV Vectors for Neuromuscular Genome Editing.

Experiment  - [In Vivo] [Small Animal Testing Center (SATC)] [Mouse]
Matched on:  -->  Ai9 SaCas9 Guide A  Ai9 SaCas9 Guide B
Heaney Jason D  Last Updated Date: 2021-03-30
 
Quantification of CRISPR/Cas editing in liver and heart following custom AAV-mediated delivery. Detection of editing in non-target tissues.

43 results for Ai9

Category Type Name Description Last Updated Date View Associated..
Model System Animal Ai9 mouse Ai9 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus.
Show Experiments (10)

Testing gRNA sequence and gRNA scaffold modified 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)
Testing AAV5 for activation of tdTomato in mouse airway
Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter)
AAV Tropism project
Cre Recombinase dose escalation study in Ai9 mice
Independent validation of Deverman delivery platform using engineered AAVs to deliver CRSIPR/Cas9 to mouse brain
Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter)
Testing AAV5 for activation of tdTomato in mouse airway club and ciliated cells
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.

Guide Ai9 SaCas9 Guide A This gRNA targets the Ai9 and related transgenes
Model System Animal Ai9 mouse (BCM) Ai9 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus.
Model System Animal Ai9-SauSpyCas9 mouse Ai9-SauSpyCas9 is a version of Ai9 that has a single guide RNA sites on both sides of the STOP cassette. This is a Cre reporter allele that has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. Ai9 mice express robust tdTomato fluorescence following Cre-mediated recombination.
Guide Ai9 SaCas9 Guide B This gRNA targets the Ai9 and related transgenes
Model System Cell Ai9 mouse immortalized fibroblasts Immortalized fibroblasts made from Ai9 (B6.Cg-Gt(ROSA)26Sor^tm9(CAG-tdTomato)Hze/J) mice
Model System Cell HEK-293T with Ai9 transient reporter assay HEK-293T cells transfected with an Ai9 inducible transgene reporter plasmid used to test gene editing activity by fluorescence. HEK293T is an epithelial-like cell that was isolated from the kidney of a patient.
Experiment In Vivo Cre Recombinase dose escalation study in Ai9 mice 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. 2021-09-16
Experiment In Vivo Testing gRNA sequence and gRNA scaffold modified in Ai9 mice. 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. 2021-04-17
Experiment In Vitro Selection of gRNA sequences and gRNA scaffold modification lead to improved editing of the Ai9 locus in vitro Reporter transgene activation by SaCas9 gRNA target and modified scaffold sequences by transient transfection in immortalized Ai9 mouse fibroblasts 2021-04-17
Experiment In Vivo Independent validation for Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9 mice 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 2021-03-30
Experiment In Vivo Comparing CRISPR/Cas9 gene editing efficiencies between AAV9 and AAVcc47 in Ai9 mice with a 1:3 Cas9 to sgRNA ratio (CMV promoter) 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. 2021-09-16
Experiment In Vivo Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 cas9 to sgRNA ratio (CMV promoter) 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. 2021-09-16
Experiment In Vivo Comparing CRISPR/Cas9 gene editing efficencies between AAV9 and AAVcc47 in Ai9 mice with a 1:1 Cas9 to sgRNA ratio (CB promoter) 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. 2021-09-16
Experiment In Vivo 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. 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. 2021-09-16
Experiment In Vivo Independent validation of Gong delivery platform using RNP-loaded nanocages to deliver CRISPR/Cas9 to mouse brain Delivery of CRISPR/Cas9 via RNP-loaded nanocages to the brain in Ai14 mice 2023-05-10
Protocol Murray-SATC_Gong-Validaiton_Gong Study Protocol Procedure for intracranial injection, immunofluorescence and imaging.
Genome Editor Cas Nuclease sNLS-SpCas9-sNLS SpCas9 with N- and C-terminal SV40 NLS
Delivery System Nanoparticle RNP-NC-RVG The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. This nanoparticle has an addition of a RVG peptide YTIWMPENPRPGTPCDIFTNSRGKRASNG which specifically interacts withthe N-acetylecholine receptor (AchR) on neuronal cells, which mediates NP entry
Antibody AB_2209751  Anti-RFP (RABBIT) Antibody
Antibody RRID:AB_2813835 
Experiment In Vivo Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain. 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. 2020-10-28
Antibody AB_2532994  Thermo-Fisher, Rat anti-GFAP
Genome Editor Cas Nuclease SauCas9
Guide Ai14 gRNA This sgRNA targets the Ai9 and related transgenes at multiple sites
Protocol Gong_Intracranial Injection Procedure for Mice Procedure for intracranial delivery to mouse brain.
Protocol Murray-SATC_Gong-Validation Brain Injection in Mice Protocol Procedure for brain injection surgical procedure, pre- and post-operative care for mice.
Antibody AB_10711040  Abcam, mouse anti-NeuN
Antibody AB_141637  Invitrogen, Donkey anti-rabbit alexa fluor 594
Antibody AB_141607  Invitrogen, Donkey anti-mouse alexafluor 488
Antibody RRID:AB_2209751 
Vector Viral Vector AAVcc47-SaCas9-Ai9 AAV2/9 expressing SaCas9 and single sgRNA under U6 promoter
Model System Animal Ai14 mouse (congenic) Ai14 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. The att site flanked neo selection cassette has been removed in this strain.
Delivery System Nanoparticle RNP-NC-CPP The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA. This nanoparticle has an addition of a cell penetrating peptide (CPP) from the TAT peptide (GRKKRRQRRRPQ) which lacks cell-type specficity
Delivery System Nanoparticle RNP-NC-no ligand The nanocapsule is a thin glutathione (GSH)-cleavable covalently crosslinked polymer coating around a preassembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and an sgRNA.
Model System Animal Ai14 mouse Ai14 mouse has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. The att site flanked neo selection cassette has been removed in this strain.
Antibody AB_2813835  Abcam, Donkey anti-rat alexa fluour 647
Antibody RRID:AB_2532994 
Antibody RRID:AB_10711040 
Antibody RRID:AB_141637 
Antibody RRID:AB_141607 
Experiment In Vivo Independent validation for Asokan Delivery Team: Evolving High Potency AAV Vectors for Neuromuscular Genome Editing. Quantification of CRISPR/Cas editing in liver and heart following custom AAV-mediated delivery. Detection of editing in non-target tissues. 2021-03-30
Protocol Heaney_SATC Tissue Processing, Imaging and Analysis Procedure for tissue preparation, imaging and analysis.