abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas9 genome editing

Abstract

Abstract Summary CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (ØXØ) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of ØXØ sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n = 97583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r ≥ 0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45–0.68). When applied to the human genome, abCRISPR generated ØXØ sequences, covering 58 875 004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing. Availability and implementation The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/