Abstract
Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are understudied. To address this gap, we developed PiCTURE, a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the PROTECTiO pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive framework for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights for the development of safer genome-editing therapies.
Competing Interest Statement
K.N. and H.O. were employed by PtBio Inc.
Footnotes
kazukinakamae{at}hiroshima-u.ac.jp; onohrms{at}hiroshima-u.ac.jp; bonohu{at}hiroshima-u.ac.jp
d233528{at}hiroshima-u.ac.jp; d246887{at}hiroshima-u.ac.jp; bonohu{at}hiroshima-u.ac.jp
yamaken37.the.answer{at}gmail.com
taro.kakuzaki{at}gmail.com
hiromasa.ono{at}gmail.com; ynaito{at}dbcls.rois.ac.jp
Abbreviations
- CBE
- cytosine base editor
- PiCTURE
- Pipeline for CRISPR-induced Transcriptome-wide Unintended RNA Editing
- PROTECTiO
- Predicting RNA Off-target compared with Tissue-specific Expression for Caring for Tissue and Organ
- RNA-seq
- RNA-sequencing
- SNP
- single-nucleotide polymorphism