Format

Send to

Choose Destination
Cell. 2018 Apr 19;173(3):665-676.e14. doi: 10.1016/j.cell.2018.02.033. Epub 2018 Mar 15.

Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors.

Author information

1
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.
2
Razavi Newman Integrative Genomics and Bioinformatics Core, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
3
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. Electronic address: patrick@salk.edu.

Abstract

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; Cas13; CasRx; RNA interference; RNA targeting; alternative splicing; frontotemporal dementia; gene editing; genome engineering; tau

PMID:
29551272
PMCID:
PMC5910255
[Available on 2019-04-19]
DOI:
10.1016/j.cell.2018.02.033

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center