CRISPRdirect is a web server designed for selecting CRISPR/Cas targets with minimal off-target sites. The CRISPR/Cas system, which uses Cas9 nuclease guided by a guide RNA (gRNA), is a powerful tool for genome engineering. CRISPRdirect performs exhaustive searches against genomic sequences to identify target sequences that meet the requirements of the 5′-NGG protospacer adjacent motif (PAM) and are specific within the genome. The server provides detailed information on target positions, sequences, and additional features such as GC content and melting temperature. It also offers a list of potential off-target sites, which can be visualized and exported in various formats. CRISPRdirect incorporates genomic sequences from multiple organisms, including human, mouse, rat, and yeast, and uses the GGGenome REST API for off-target searches, allowing for mismatches and gaps. The server is freely available at http://crispr.dbcls.jp/ and can be used for automated gRNA design on a genome-wide scale.CRISPRdirect is a web server designed for selecting CRISPR/Cas targets with minimal off-target sites. The CRISPR/Cas system, which uses Cas9 nuclease guided by a guide RNA (gRNA), is a powerful tool for genome engineering. CRISPRdirect performs exhaustive searches against genomic sequences to identify target sequences that meet the requirements of the 5′-NGG protospacer adjacent motif (PAM) and are specific within the genome. The server provides detailed information on target positions, sequences, and additional features such as GC content and melting temperature. It also offers a list of potential off-target sites, which can be visualized and exported in various formats. CRISPRdirect incorporates genomic sequences from multiple organisms, including human, mouse, rat, and yeast, and uses the GGGenome REST API for off-target searches, allowing for mismatches and gaps. The server is freely available at http://crispr.dbcls.jp/ and can be used for automated gRNA design on a genome-wide scale.