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Project: Enhancing CRISPR Gene Editing in Somatic Tissues by Chemical Modification of Guides and Donors

PI:   Erik J Sontheimer, PhD  NIH Report

RNA-guided CRISPR genome editing systems promise to revolutionize the treatment of inherited disease. Safe, effective, and target-tissue-specific delivery of the guide RNA that directs editing is a critical hurdle in the development of clinical applications for engineered CRISPR systems. Using strategies validated for the delivery of other categories of nucleic acid therapeutics, we have established a framework for complete chemical modification of CRISPR guides, thereby conferring in vivo stability and effective biodistribution properties. The proposed research will optimize these guides, as well as other editing components, for clinical use.
Summary of data submissions:
  • Data for 10  experiments were submitted on 2021-04-15 SCGE ID:1030
  • Data for 1  experiments were submitted on 2021-10-01 SCGE ID:1052
  • 1 of 11 experiments have been validated

Submissions Details

SCGE ID:1030 - Submission Date: 2021-04-15 
Experiment Name Type Description
Testing newly chemically modified crRNA and tracrRNA in mTmG mouse embryonic fibroblasts

 In Vitro 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.
Testing newly chemically modified crRNA and tracrRNA in mouse Hepa 1-6 cells

 In Vitro Chemically modified crRNA and tracrRNA were delivered by electroporation to mouse Hepa 1-6 cells. Editing activity was determined by Sanger sequencing
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

In Vivo Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged.
Delivery of unmodified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain

In Vivo Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged.
Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain

In Vivo Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged.
Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, extended PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain

In Vivo Chemically modified crRNA and tracrRNA were injected into the intra-striatum of mTmG reporter mice and activation of GFP expression was imaged.
Testing newly chemically modified crRNA and tracrRNA to activate the TLR reporter in human cells

 In Vitro 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.
Testing newly chemically modified crRNA and tracrRNA to activate the TLR1 reporter in human cells

 In Vitro 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.
Testing newly chemically modified crRNA and tracrRNA in mouse Neuro 2A cells

 In Vitro Chemically modified crRNA and tracrRNA were delivered by electroporation to mouse Neuro2A cells. Editing activity was determined by Sanger sequencing
Delivery of RNP containing chemically modified crRNA C20 with chemically modified tracrRNA T2-PS to determine the RNP distribution in TLR-MCV mouse brain

 In Vivo Chemically modified crRNA and tracrRNA were injected into the striatum of TLR-MCV mice and the distribution of RNP was imaged.
SCGE ID:1052 - Submission Date: 2021-10-01 
Experiment Name Type Description
Testing preparation for independent validation at The Jackson Laboratory Small Animal Testing Center

 In Vivo Delivery of chemically modified, phosphorothioate (PS)-stabilized crRNA with chemically modified, PS-stabilized tracrRNA to activate the mTmG reporter in mouse brain