**5. MultiSite Gateway technology for donor DNA plasmid construction**

The original Gateway cloning system was further improved for cloning multiple DNA fragments into a single vector [28, 62]. The improved cloning method is called MultiSite Gateway[29, 63] (**Figure 5**). In the MultiSite Gateway cloning method, numerous attB, attP, attL, and attR- variant sites were developed. Each attB site is specifically recombined with the corresponding- attP site by the BP reaction, to generate the corresponding attL and attR sites. Similarly, each attL-

**Figure 5.** Cloning four-fragments by the MultiSite Gateway method. The strategy for the construction of the donor DNA plasmid is shown.-

site is specifically recombined with the corresponding attR site by the LR reaction. At present,- up to four DNA fragments can be cloned into a single vector. The 4-fragment MultiSite Gateway- cloning technology is suitable for the construction of a donor DNA plasmid for CRISPR/Cas9 and HR-mediated gene knock-in (**Figure 5**). In the first step of the 4-fragment cloning, each- PCR-amplified DNA fragment is cloned into the corresponding pDONR vectors. As shown- in **Figure 5**, the orientation of the att site sequence is important. The attB3r, attB4r, and attB5r- sequences are reversely oriented relative to the attB3, attB4, and attB5 sequences, respectively.- In the second cloning step, the four entry clones are mixed with an appropriate destination vector and are subjected to the LR reaction. Thus, the four DNA fragments are simultaneously- assembled and inserted into a single destination vector, in the desired order and orientation.- The outline of our example is described below [52].-

We used a CRISPR/Cas9-mediated gene knock-in, in order to express the wild type or mutant gene of interest (human RAD52 gene in our case) with its own native promoter in the genome and to examine the cellular effects of the mutant protein expressed at the endogenous level. The 1000 bp genomic DNA region upstream from the start codon of the gene of interest was amplified by PCR with primer sets containing attB1 or attB5r sequences at the 5′ ends and was cloned between the corresponding attP1 and attP5r sites of the first pDONR vector. The 1000 bp genomic DNA region downstream from the stop codon of the gene was cloned between the attP3 and attP2 sites of the fourth pDONR vector (**Figures 3** and **5**). The cDNA of the gene (wild type or mutant) was first cloned via standard Gateway cloning into a destination vector (pT-Rex-DEST30), for gene expression with the CMV promoter of the vector (**Figure 4**). In the destination clone, the SV40 polyadenylation region required for transcriptional termination is present downstream from the stop codon of the cloned gene. We amplified the DNA region encoding the cDNA of the gene and the SV40 polyadenylation region by PCR, using the destination clone as the PCR template. The PCR product was cloned between the attP5 and attP4 sites of the second pDONR vector (**Figure 5**). The DNA region containing the SV40 promoter, neomycin resistance gene, and polyadenylation region was amplified by PCR with pT-Rex-DEST30 as the PCR template and was cloned between the attP4r and attP3r sites of the third pDONR vector (**Figure 5**). The DNA fragments in each of the entry clones were verified by DNA sequencing. Then, the LR reaction was performed with the confirmed four types of entry clones and the destination vector (a simple vector, pDEST14, in our case). Thus, the complicated donor DNA plasmids could be constructed relatively easily, by using the MultiSite Gateway technology.-
