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Motivation The CRISPR/Cas9 system is widely used for genome editing. The editing efficiency of CRISPR/Cas9 is mainly determined by the guide RNA (gRNA). Although many computational algorithms have been developed in recent years, it is still a challenge to select optimal bioinformatics tools for gRNA design in different experimental settings. Results We performed a comprehensive comparison analysis of 15 public algorithms for gRNA design, using 16 experimental gRNA datasets. Based on this analysis, we identified the top-performing algorithms, with which we further implemented various computational strategies to build ensemble models for performance improvement. Validation analysis indicates that the new ensemble model had improved performance over any individual algorithm alone at predicting gRNA efficacy under various experimental conditions. Availability and implementation The new sgRNA design tool is freely accessible as a web application via https://crisprdb.org. The source code and stand-alone version is available at Figshare (https://doi.org/10.6084/m9.figshare.21295863) and Github (https://github.com/wang-lab/CRISPRDB). Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]