The powerful CRISPR-Cas9 genome editing system is hindered by its off-target effects. Although in silico tools have been developed for single-strand guide RNA (sgRNA) design and CRISPR off-target evaluation, the genome-wide CRISPR off-target prediction performance is limited. In this study, we proposed that the modelling of molecular interactions within the Cas9-sgRNA-DNA complex can substantially improve the genome-wide off-target prediction. To this end, we systematically performed molecular dynamics (MD) simulations to capture the molecular interaction features of the CRISPR-Cas9 system. CRISOT, which is a powerful tool package suite that contains various modules for genome-wide CRISPR off-target evaluation and sgRNA optimization, is presented.