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In Vitro
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Validating Traffic Light Reporter 7 (TLR7) Mouse Model for NHEJ in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Traffic Light Reporter 7 (TLR7) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides 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, 169. Total blast analyzed by Sanger sequencing, 117. |
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2024-04-05
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In Vitro
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Validating Traffic Light Reporter 7 (TLR7) Mouse Model for HDR in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Traffic Light Reporter 7 (TLR7) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides and HDR donor template, 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, 103. Total blast analyzed by Sanger sequencing, 64. |
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2024-04-05
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In Vitro
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Validating Gene Editing Reporter 12 (GER12) Mouse Model in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Gene Editing Reporter 12 (GER12) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides 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, 58-69. Total blast analyzed by Sanger sequencing, 51-52. |
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2024-02-23
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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 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 Vitro
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Validating Traffic Light Reporter 2 (TLR2) Mouse Model in Heterozygous Blastocysts using SaCas9
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WT female mouse and homozygous male mouse containing the Traffic Light Reporter 2 (TLR2) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for tagRFP 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: Total blasts analyzed expressing the fluorescent reporter, 55-67. Total blast analyzed by Sanger sequencing, 55-67. |
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2023-09-27
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In Vitro
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Validating Traffic Light Reporter 2 (TLR2) Mouse Model in Heterozygous Blastocysts using SpCas9
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WT female mouse and homozygous male mouse containing the Traffic Light Reporter 2 (TLR2) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for tagRFP 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, 46-120. Total blast analyzed by Sanger sequencing, 8-64. |
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2023-09-27
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In Vitro
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Validating Gene Editing Reporter 19 (GER19) Mouse Model in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Gene Editing Reporter 19 (GER19) reporter driven by the CAG promoter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with two guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for GFP fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced to determine efficiency of deleting the IVS2-654 splicing variant. 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, 53. Total blast analyzed by Sanger sequencing, 48. |
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2023-09-27
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In Vitro
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Validating Gene Editing Reporter 10 (GER10) Mouse Model in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Gene Editing Reporter 10 (GER10) reproter driven by the CAG promoter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Adenine Base Editor RNP complex and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for Venus (GFP) fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced to determine efficiency of base changes at the target region. 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, 174. Total blast analyzed by Sanger sequencing, 112. |
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2023-09-27
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In Vitro
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Validating Gene Editing Reporter 18 (GER18) Mouse Model in Heterozygous Blastocysts
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WT female mouse and homozygous male mouse containing the Gene Editing Reporter 18 (GER18) reporter driven by the EF-1 alpha promoter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with two guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for GFP fluorescence and scored for fluorescence signal. Blastocysts were then PCR amplified for the reporter allele and Sanger sequenced to determine efficiency of deleting the IVS2-654 splicing variant. 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, 61. Total blast analyzed by Sanger sequencing, 54. |
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2023-09-27
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In Vitro
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Validating Traffic Light Reporter 2 (TLR2) Mouse Model in Homozygous Blastocysts using SpCas9
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Male and female homozygous mice mouse containing the Traffic Light Reporter 2 (TLR2) reporter at the Rosa26 locus were mated. Females were super-ovulated and zygotes harvested. Zyotes were electroporated with Cas9 RNP along with test guides and were cultured 96 hours to blastocyst stage. Blastocysts were imaged for tagRFP 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, 156-193. Total blast analyzed by Sanger sequencing, 84-120. |
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2023-09-27
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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 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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[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]
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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[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 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 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 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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