Computational Molecular Biology 2025, Vol.15, No.2, 102-111 http://bioscipublisher.com/index.php/cmb 10 7 methods represent the future development direction, but how to strike a balance among transparency, interpretability and prediction accuracy remains an urgent problem to be solved in this field. 7 Predictive Analysis of Human EMX1 Gene Off-target 7.1 Research background of EMX1 gene editing The EMX1 gene (Empty Spiracles Homeobox 1) is an important transcription factor related to human development and is often selected as the target model for CRISPR gene editing research. Due to its significant role in the development of the nervous system, researchers pay particular attention to off-target effects when using CRISPR-Cas9 to knockout EMX1. Early studies have shown that even well-designed Sgrnas have potentially dozens of approximate targets in the human genome. Therefore, this gene has become a typical case in off-target prediction and validation studies (Figure 2) (Chaudhari et al., 2020). Figure 2 (A) Overlap among three replicates for sites nominated by GUIDE-seq, CIRCLE-seq, and SITE-seq across all eight gRNAs. (B) Correlation of read counts for nominated sites between two replicates for GUIDE-seq (R2 = 0.54–0.67), CIRCLE-seq (R2 = 0.80–0.83), and SITE-seq (R2 = 0.90–0.91) read counts. The plots present data for replicates 1 and 2, and the R2 range is calculated from all three comparisons (1 vs. 2, 2 vs. 3, 1 vs. 3). (C) Distributions of read counts for nominated sites obtained by for GUIDE-seq, CIRCLE-seq, and SITE-seq grouped by the number of mismatches and gaps in the site with respect to gRNA sequence. gRNA, guide RNA (Adopted from Chaudhari et al., 2020)
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