**1. Introduction**

Genome editing has been an important technique to investigate gene function in biology since- before the development of the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. In microorganisms such as *Escherichia coli (E. coli)* and yeast, gene disruption can be achieved by simply introducing a donor DNA into the cells without inducing a DSB in- the targeted gene [1–3]. With regards to gene disruption in vertebrate cells, chicken B lymphocyte-

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line DT 40 cells have been widely used because of their high efficiency of homologous DNA- recombination [4–6]. Similar to *E. coli* and yeast cells, gene disruption in DT 40 cells can also be achieved by introducing a DNA vector for gene targeting into the cells. In contrast, gene disruption by this method in human cells is difficult, because of the inefficient homologous recombination in the cells. Therefore, gene knockdown by RNA interference (RNAi) has usually been used- in human cells, in order to examine the functions of human genes [7]. However, the targeted- protein cannot be completely eliminated from the cells by RNAi. Therefore, an efficient method- for targeted gene disruption in human cells has been keenly desired.-

The CRISPR/Cas9 method is an innovative genome editing technology. The history of this technology originated from the finding of unusual, functionally unknown repeated sequences in *E. coli* [8, 9]. In the repeated sequences, highly homologous repeats are separated by nonrepetitive nucleotides as spacers. Repeated sequences, composed of repeats and spacers, were also- found in numerous genomes of other bacteria and archaea [10] and were named CRISPR [11].- In addition, well-conserved genes were identified adjacent to the CRISPR loci and were named- CRISPR-associated (Cas) genes [11]. Some of the Cas protein families were found to share- sequence homology with proteins involved in DNA metabolism, such as helicases and exonucleases [11], and the purified Cas proteins exhibited endonuclease activity [12–14]. Sequences- identical to the CRISPR spacers were found among bacterial mobile genetic elements, such as plasmids and phages, suggesting that the CRISPR spacers in the bacterial genome are derived- from DNA fragments of the invading foreign genetic elements [15, 16]. In bacteria, CRISPR/Cas- serves as a defense system against the invasion of mobile genetic elements [17]. The molecular mechanism of the defense system was elucidated by biochemical experiments [13, 14, 18, 19]. Cas proteins bind to RNA transcribed from the CRISPR spacer sequence and cleave the- precursor CRISPR RNA (pre-crRNA) [18]. Cas proteins exist in the complex with the cleaved- mature crRNA [18] and cleave invading foreign genetic elements mediated by the crRNA guide,- containing the complementary sequence with the targeted genetic elements [13, 14, 19]. Among- the Cas family proteins, a single Cas9 protein complexed with a crRNA can introduce a specific- DSB at the desired site in the target DNA [13, 14]. Therefore, the CRISPR/Cas9 system is being- applied for genome engineering [20, 21]. The biology and technology of the CRISPR/Cas system- are described in detail in excellent reviews [22–27].-

A variety of CRISPR/Cas9-mediated genome editing tools is now commercially available. A- gene knock-out can be accomplished simply by using the CRISPR/Cas9 tool. However, for a- gene knock-in or replacement, a donor DNA must also be prepared individually. Here, we- describe the usefulness of the MultiSite Gateway technology [28, 29] for the construction of the donor DNA plasmid.-
