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

• Published in: Cell reports (Cambridge) 2020-11, Vol.33 (5), p.108350-108350, Article 108350
• Main Authors: Marina, Ryan J., Brannan, Kristopher W., Dong, Kevin D., Yee, Brian A., Yeo, Gene W.
• Format: Article
• Language: English
• Subjects: ADAR2; Adenosine Deaminase - metabolism; Base Sequence; Cas13; Cas9; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Genetic Engineering; HEK293 Cells; Humans; RNA editing; RNA Editing - genetics; RNA targeting Cas9; RNA, Guide, CRISPR-Cas Systems - metabolism; RNA-Binding Proteins - metabolism; RNA-Seq; spacerless guide RNA; Transcriptome - genetics; transcriptome engineering
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2020.108350

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. [Display omitted] •RNA-targeting Cas9-ADAR fusions direct specific A-to-I editing with modified gRNA•Cas9-ADAR targets exogenous and endogenous transcripts in a spacer-independent manner•Orientation of Cas9- and Cas13-ADAR fusions influences on-target efficacy•Cas-based ADAR strategies have distinct transcriptome-wide off-target edits CRISPR-mediated transcriptome editing is an alternative strategy for genetic engineering. Marina et al. introduce a Cas9-directed method for RNA editing, which can be performed with a spacer-lacking guide RNA (gRNA). This system is comparable to other RNA-targeting Cas platforms both in on- and off-target capability.