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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Animals
  • Bacterial Proteins / metabolism
  • CRISPR-Cas Systems*
  • Cell Differentiation
  • Computational Biology / methods*
  • Escherichia coli / metabolism
  • Gene Expression Profiling
  • Genetic Engineering / methods*
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Lentivirus / genetics
  • Mice
  • Protein Engineering / methods*
  • RNA / analysis*
  • RNA Interference
  • RNA, Guide / genetics
  • Ruminococcus
  • Sequence Analysis, RNA
  • Transcriptome

Substances

  • Bacterial Proteins
  • RNA, Guide
  • RNA