Experiment: Testing newly chemically modified crRNA and tracrRNA to activate the TLR reporter in human cells
PI: Erik J Sontheimer, PhD
Description: 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.
Editing Assay:
Calculated editing efficiency by flow cytometry analysis of RFP expression.
Parent Project: Enhancing CRISPR Gene Editing in Somatic Tissues by Chemical Modification of Guides and Donors
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Other experiments in this project: 10
- Testing newly chemically modified crRNA and tracrRNA in mTmG mouse embryonic fibroblasts
- Testing newly chemically modified crRNA and tracrRNA in mouse Hepa 1-6 cells
- 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
- Delivery of unmodified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
- Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
- Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain
- Testing newly chemically modified crRNA and tracrRNA to activate the TLR1 reporter in human cells
- Testing newly chemically modified crRNA and tracrRNA in mouse Neuro 2A cells
- Delivery of RNP containing chemically modified crRNA C20 with chemically modified tracrRNA T2-PS to determine the RNP distribution in TLR-MCV mouse brain
- Testing preparation for independent validation at The Jackson Laboratory Small Animal Testing Center
Download: Submitted files
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Results |
| Editing Efficiency | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Condition | Cell Type | Sex | Editor | Model | Delivery | Target Locus | Guide | Dosage | % of RFP+ cells | Image |
| STS118 (TLR-MCV1a) (5 pmol RNP) | Female | SpyCas9-3xNLS | HEK-293T-disrupted_GFP with MCV-mcherry-Puro | FSD117D1 | TLR MCV reporter | STS118 (TLR-MCV1a; Sp_t2:Sp_c20_TLR_MCV1a) | 5 pmol RNP | |||
| STS118 (TLR-MCV1a) (20 pmol RNP) | Female | SpyCas9-3xNLS | HEK-293T-disrupted_GFP with MCV-mcherry-Puro | FSD117D1 | TLR MCV reporter | STS118 (TLR-MCV1a; Sp_t2:Sp_c20_TLR_MCV1a) | 20 pmol RNP | |||
| STS119 (TLR-MCV1b) (20 pmol RNP) | Female | SpyCas9-3xNLS | HEK-293T-disrupted_GFP with MCV-mcherry-Puro | TLR MCV reporter | STS119 (TLR-MCV1b; Sp_t2:Sp_c20_TLR_MCV1b) | 20 pmol RNP | ||||
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