Targeted Trans-splicing Using CRISPR-Cas Systems
This technology provides a novel method of targeting ribonucleic acids for trans-splicing using any CRISPR/Cas13 orthologs having two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) endonuclease domains (Type VI CRISPR effectors). The method uses the principles of endogenous RNA processing and RNA-writing using a CRISPR/Cas13 protein to initiate the targeted trans-splicing of defective pre-mRNAs, effectively correcting mutations at the pre-translation level in the nucleus and allowing for the treatment of essentially all types of mongenic diseases. The system is a significant improvement over CRISPR-mediated genomic DNA editing and other gene therapies as it allows for the option of transient RNA repair while improving both the specificity and the efficiency of trans-splicing events.
Researchers
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targeted trans-splicing using crispr/cas13
United States of America | Granted | 11,767,528 -
targeted trans-splicing using crispr/cas13
Australia | Pending -
targeted trans-splicing using crispr/cas13
Canada | Published application -
targeted trans-splicing using crispr/cas13
European Patent Convention | Published application -
targeted trans-splicing using crispr/cas13
Japan | Published application -
targeted trans-splicing using crispr/cas13
Korea (south) | Published application -
targeted trans-splicing using crispr/cas13
United States of America | Published application
Technology
The CRISPR/Cas13 system (a complex of the nuclease-inactive Cas13, guide RNA, and a repair splice donor/acceptor template), specifically recognizes target sequences on the defective pre-mRNA. The system then mediates the process of trans-splicing, utilizing the repair template to join exons within the pre-mRNA or replace targeted exons. This precise modification ensures the desired edits within the repair template are incorporated into the final mRNA. As a result, a modified RNA molecule is produced, which can encode the corrected protein, a novel protein or serve as therapeutic noncoding or regulatory RNA.
Problem Addressed
This technology addresses the limitations of traditional RNA splicing methods by improving the efficiency and specificity of trans-splicing. It offers a mechanism to precisely repair RNA, increasing protein diversity without altering the genomic DNA. Additionally, it can be exploited for molecular therapy to correct mutated gene products and treat a wide range of diseases.
Advantages
- The use of Cas13 and guide RNA allows for precise manipulation of RNA transcripts
- Enables exon swapping at 5’/3’ ends and internally, in dividing and post-mitotic cells
- Trans-splicing at the pre-mRNA level does not cause changes to genomic DNA
- Potential for one off-the-shelf therapeutic formulation to be able to correct multiple different disease-causing mutations in a gene across a patient population
Publications
Borrajo, J., et al. "Programmable Multi-Kilobase RNA Editing Using CRISPR-Mediated Trans-Splicing." bioRxiv (2023). https://doi.org/10.1101/2023.08.18.553620.
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