**9. Conclusion**

The CRISPR/Cas9 technology has opened a new window to investigate gene functions by targeted knock-ins. By replacing the endogenous gene with the mutant gene, the cellular effects of the mutant can be examined under the most suitable native conditions of the gene expressed from the native promoter. The native expression level of the gene is also important for investigatingthe intracellular localization and behavior of the gene product, because overexpression of the gene by a nonnative promoter sometimes induces artifactual effects on the intracellular localization of the protein. The CRISPR/Cas9-mediated knock-in of specific tag sequences into the endogenous gene allows the investigation of the intracellular localization of the protein at the native expression level, by monitoring the introduced tag sequences. The construction of a complicated donor DNA is required for gene knock-in mediated by HR.-This is a bottleneck point for the CRISPR/Cas9-mediated targeted knock-in technology. Standard Gateway cloning is a popular method for constructing ordinary expression plasmids and is therefore more commonly used as compared to MultiSite Gateway cloning. However, MultiSite Gateway cloning is a quite useful method, especially for constructing the complicated donor DNA plasmid. Therefore, this technology will contribute to the spread of CRISPR/Cas9-mediated targeted knock-in methods.-
