Validation Experiment: Independent validation of Sontheimer delivery platform using heavily modified guide RNAs complexed with Cas9 proteins to deliver CRISPR/Cas9 to mouse brain
PI: Stephen A Murray, PhD
Description: 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.
Editing Assay:
Editing efficiency calculated by counting eGFP expressing cells (DAB+) 0.5 mm from either side of the injection track compared to hematoxylin labeled nuclei in mouse striatum. Cell counting is done with FIJI macro. #DAB+ (eGFP+) cells/total number of cells in ROI *100 = Percent edited cells in ROI
Parent Project: The Jackson Laboratory Gene Editing Testing Center - Sontheimer Validation (Sontheimer, Eric)
Download: Submitted files
Original Experiment/s that are being validated:
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Results |
| Editing Efficiency | ||||||||||
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| Condition | Tissue | Sex | Editor | Model | Target Locus | Guide | Dosage | Injection Frequency | % edited cells in ROI | Image |
| DNMT1 C20/T2 SpyCas9 Males | striatum (TARGET) | Male | SpyCas9-3xNLS | mTmG mouse (congenic) | Dnmt1 | STS204 (DNMT1; Sp_t2:Sp_c20_Dnmt1) | 100 uM | once |
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| DNMT1 C20/T2 SpyCas9 Females | striatum (TARGET) | Female | SpyCas9-3xNLS | mTmG mouse (congenic) | Dnmt1 | STS204 (DNMT1; Sp_t2:Sp_c20_Dnmt1) | 100 uM | once |
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| mTmG c20/T2 SpyCas9 Males | striatum (TARGET) | Male | SpyCas9-3xNLS | mTmG mouse (congenic) | mTmG reporter transgene | STS159 (mTmG; Sp_t2:Sp_c20_mTmG) | 100 uM | once |
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| mTmG c20/T2 SpyCas9 Females | striatum (TARGET) | Female | SpyCas9-3xNLS | mTmG mouse (congenic) | mTmG reporter transgene | STS159 (mTmG; Sp_t2:Sp_c20_mTmG) | 100 uM | once |
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Experiment Wide Protocols |
| Title | Description | File Download | SCGE ID |
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