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Schematic representation of the sequence design process for the 180k synthetic gRNA-off-target library. The 180k library included 184,561 carefully designed off-target sequences. These sequences included: 89,730 single-mutation off-targets, which could be further divided into single mismatches (OFF_Mis), single deletions (OFF_Del), and single insertions (OFF_Ins); 93,579 off-targets with multiple mismatches (OFF_Mul), which were generated via a traversal strategy and a predictive strategy; and 1252 pairs collected from computational predictions or experimental assays (e.g., GUIDE-seq). The former two groups were utilized to quantify indel frequencies associated with different off-target types at a large scale. The latter group was used to validate our method.

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