Reprogrammed tracrRNA enables Cas9-mediated RNA editing and trans-splicing
This dataset contains next-generation sequencing data generated to characterize RETREAT, a Cas9-based RNA targeting platform guided by reprogrammed tracrRNAs (Rptrs). Three types of sequencing data are included. Targeted amplicon sequencing of endogenous transcripts. Amplicon NGS was performed on HEK293T cells expressing RETREAT or REPAIR-v1 constructs targeting ACTB, KRAS, and CYFIP2. These data quantify on-target A-to-I(G) RNA editing efficiencies and bystander edits at endogenous sites. Transcriptome-wide mRNA sequencing for off-target analysis. PolyA-enriched mRNA-seq libraries were generated from cells expressing RETREAT or REPAIR-v1 with targeting or non-targeting guides. These datasets support global profiling of A-to-I(G) edits, comparison of transcriptome-wide off-target activity, and measurement of editor and target transcript abundance. Targeted sequencing of 3-prime and 5-prime trans-splicing reporters. Amplicon NGS was used to quantify splice junctions in engineered 3-prime and 5-prime split-EGFP reporters in the presence or absence of Rptr-guided dCas9. These data assess how RETREAT influences splice-site choice and RNA junction repair. Together, these sequencing datasets support analyses of on-target RNA editing, transcriptome-wide off-target activity, and programmable RNA trans-splicing using Rptr-guided Cas9.