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Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Affiliations
- Razavi Newman Integrative Genomics and Bioinformatics Core, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Correspondence
- Corresponding author
Affiliations
- Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
- Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
Correspondence
- Corresponding author
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Article Info
Highlights
- •Computational pipeline identifies the RNA-targeting type VI-D CRISPR-Cas family
- •Ortholog screen and protein engineering yields the programmable ribonuclease CasRx
- •CasRx RNA knockdown exhibits favorable efficiency and specificity relative to RNAi
- •Neuronal AAV delivery of dCasRx splice effectors alleviates tau mis-splicing
Summary
Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.
Keywords:
CRISPR, genome engineering, gene editing, RNA targeting, RNA interference, alternative splicing, frontotemporal dementia, tau, Cas13, CasRxTo access this article, please choose from the options below
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