Systematic identification and characterization of high efficiency Cas9 guide RNAs for therapeutic targeting of ADAR
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ABSTRACT:
PROVIDER: PRJNA1179362 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31146738_1.fastq.gz | Fastqsanger.gz | |||
| SRR31146738_2.fastq.gz | Fastqsanger.gz | |||
| SRR31146739_1.fastq.gz | Fastqsanger.gz | |||
| SRR31146739_2.fastq.gz | Fastqsanger.gz | |||
| SRR31146740_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
2025-01-08 | GSE280530 | GEO
Project description:RNA base editing represents a promising alternative for genome editing. Recent approaches harness the endogenous RNA editing enzyme ADAR to circumvent problems related to the ectopic expression of an editing enzyme, but they suffer from sequence restriction, lack of efficiency, and bystander editing. Here, we present in‐silico optimized CLUSTER guide RNAs, which bind their target mRNAs in a multivalent fashion and thereby enable editing with unprecedented precision as shown by next generation sequencing. CLUSTER guide RNAs can be genetically encoded and manufactured into viruses to work in various cell lines. They achieve on‐target editing on endogenous transcripts like GUSB and NUP43 with yields up to 45% without bystander editing and have been shown to recruit endogenous ADAR in vivo. The CLUSTER approach tremendously enlarges the sequence space available for guide RNA design and opens new avenues for drug development in the field of RNA base editing.
2021-09-20 | GSE184244 | GEO
Project description: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.
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Project description:Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR/Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. We leverage our TRIP technology to integrate ~1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5′ overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.
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