Abstract Motivation: Despite the growing popularity in using CRISPR/Cas9 technology for genome editing and gene knockout, its performance still relies on well-designed single guide RNAs (sgRNA). In this study, we propose a web application for the Design and Optimization (CRISPR-DO) of guide sequences that target both coding and non-coding regions in spCas9 CRISPR system across human, mouse, zebrafish, fly and worm genomes. CRISPR-DO uses a computational sequence model to predict sgRNA efficiency, and employs a specificity scoring function to evaluate the potential of off-target effect. It also provides information on functional conservation of target sequences, as well as the overlaps with exons, putative regulatory sequences and single-nucleotide polymorphisms (SNPs). The web application has a user-friendly genome–browser interface to facilitate the selection of the best target DNA sequences for experimental design. Availability and Implementation: CRISPR-DO is available at http://cistrome.org/crispr/ Contact: qiliu@tongji.edu.cn or hanxu@jimmy.harvard.edu or xsliu@jimmy.harvard.edu Supplementary information: Supplementary data are available at Bioinformatics online.