BioProject

In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. More...

In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. Targeted RNA editing technologies hold great process in the area of post-transcriptional genomic engineering and impermanent therapeutic correction of disease-causing variants, though off-target editing events still remain a concern. Here, we perform paired-end poly(A)+ purified RNA-seq to characterize transcriptome-wide in HEK293T cells transfected with catalytically-dead Cas9-, Cas13b, or Cas13d fused to human ADAR2 deaminase domain containing a hyperactive editing mutation (E488Q) along with either CYFIP2¬-targeting or non-targeting gRNA, in duplicate. We find that all technologies tested introduced widespread but distinct off-target mutations in the transcriptome, with the majority of edits occurring in 3’UTR and CDS regions of genes. Many of these edits, however, were in transcripts with low coverage, with the majority of editing rates being below 20%. This study is the first to characterize Cas-based RNA editing technologies in a direct manner, and reveals the relative similarity of currently available methods both in terms of on-target and off-target efficacy. Overall design: Examination of mRNA profiles of HEK293T cells 48 hours after transfection with dCas9-, dCas13b, or dCas13d-ADAR2(E488Q) fusions with either CYFIP2¬-targeting or NT gRNA Less...