Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing

Cell Rep. 2020 Nov 3;33(5):108350. doi: 10.1016/j.celrep.2020.108350.

Abstract

Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.

Keywords: ADAR2; CRISPR; Cas13; Cas9; RNA editing; RNA targeting Cas9; spacerless guide RNA; transcriptome engineering.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Deaminase / metabolism
  • Base Sequence
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • Genetic Engineering*
  • HEK293 Cells
  • Humans
  • RNA Editing / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • RNA-Binding Proteins / metabolism
  • RNA-Seq
  • Transcriptome / genetics

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • RNA-Binding Proteins
  • CRISPR-Associated Protein 9
  • ADARB1 protein, human
  • Adenosine Deaminase