CRISPR Cas9 and other sequence-specific endonucleases are fundamental genome editors supporting gene knockout and gene therapy. A speedy and accurate computational allele designer is required for a high through-put gene mutagenesis pipeline using these new techniques. An automatic system, Cas9 online designer (COD), was created to screen Cas9 targets and off-targets, as well as to provide gene knockout and genotyping strategies. A gene knockout rat model was successfully created and genotyped under the direction of this online system confirming its ability to predict real targets and off-targets. Gene knockout strategies to mutate 72 rat cytochrome P450 genes were designed instantly by the system to demonstrate its high-throughput efficiency. Also, the system used an off-target scoring matrix which can be applied to any sequence-specific genome editing tools besides Cas9. The COD system (http://cas9.wicp.net) has established a speedy, accurate, flexible and high through-put computational gene knockout pipeline supporting the sequence-specific endonuclease induced mutagenesis.