Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors

By Silvana Konermann, Peter Lotfy, Nicholas J. Brideau, Jennifer Oki, M. Shokhirev, P. Hsu. Cell, 2018

1,022 citations recorded by the source corpus.

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 which 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.

Subfields: Cardiology and Cardiovascular Medicine, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/6011ef724bab1a601e76fc3f8625dfb8457085c4

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In Molecular Biology

1. Benjamin J. Blencowe
2. Tessa G. Montague
3. Eivind Valen
4. Jernej Ule

Citations in the corpus

• Alternative Splicing Regulatory Networks: Functions, Mechanisms, and Evolution. (2019, background): “The resulting information will facilitate further development of strategies for the therapeutic targeting of the splicing process, for example, through the use of small molecules, antisense oligonucleotides, and CRISPR-based approaches for editing cis elements or modulating splicing regulation (Fong”
• CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing (2019, background): “For instance, CRISPR–Cas13 has been harnessed for transcript knockdown (25), live-cell transcript imaging (14) and RNA base editing (13).”
• CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “Upon further engineering of this effector, CasRx was developed for efficient RNA-guided RNA targeting in human cells (66, 67).”