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Systematic identification and characterization of high efficiency Cas9 guide RNAs for therapeutic targeting of ADAR
Benjamin G Gowen1, Kory Melton1, Weng In Leong1
1Spotlight Therapeutics, Hayward, California, United States of America.
Abstract:
Therapeutic targeting of the adenosine deaminase ADAR has great potential in cancer and other indications; however, it remains unclear what approach can enable effective and selective therapeutic inhibition. Herein, we conduct multi-staged guide RNA screening and identify high efficiency Cas9 guide RNAs to enable a CRISPR/Cas-based approach for ADAR knockout. Through characterization in human primary immune cell systems we observe similar activity with two-part guide RNA and single guide RNA, dose responsive activity, similar guide activity rank order across different cell types, and favorable computational off-target profiles of candidate guide RNAs. We determine that knockout of ADAR using these guide RNAs induces pharmacodynamic responses primarily consisting of immunological responses such as a type I interferon response, consistent with the known function of ADAR as a key regulator of dsRNA sensing. We observe similar biological effects with targeting only the p150 isoform or both p110 and p150 isoforms of ADAR, indicating that at least in the contexts evaluated, loss of p150 ADAR mediates the primary response. These findings provide a resource of well-characterized, high efficiency ADAR-targeting Cas9 guide RNAs suitable for genomic medicines utilizing different delivery modalities and addressing different therapeutic areas.
Insights
Researchers developed CRISPR/Cas9 guide RNAs for adenosine deaminase (ADAR) knockout, inducing immune responses. This offers a new avenue for genomic medicine targeting ADAR in diseases like cancer.
Area of Science:
- Genomic Medicine
- Molecular Biology
- Immunology
Background:
- Adenosine deaminase (ADAR) is a therapeutic target with potential in cancer and other diseases.
- Effective and selective inhibition strategies for ADAR remain unclear.
- CRISPR/Cas9 technology offers a precise method for gene editing.
Purpose of the Study:
- To identify high-efficiency Cas9 guide RNAs for ADAR knockout using CRISPR/Cas9.
- To characterize the pharmacodynamic and biological responses following ADAR knockout.
- To evaluate the role of different ADAR isoforms in cellular responses.
Main Methods:
- Multi-staged guide RNA screening for ADAR targeting.
- Characterization of guide RNA activity in human primary immune cells.
- Assessment of dose-responsive activity and off-target profiles.
- Analysis of immunological responses, including type I interferon induction.
Main Results:
- Identified high-efficiency Cas9 guide RNAs for ADAR knockout.
- Observed dose-responsive activity and favorable computational off-target profiles.
- ADAR knockout induced significant immunological responses, including type I interferon.
- Targeting the p150 isoform of ADAR was sufficient to mediate the primary biological response.
Conclusions:
- Developed a resource of well-characterized, high-efficiency ADAR-targeting guide RNAs for CRISPR/Cas9 applications.
- ADAR knockout elicits immunological responses, highlighting its role in dsRNA sensing.
- The p150 isoform of ADAR appears to be the primary mediator of observed biological effects.
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