**7. Strategies and improvements for generating knock-in cells**

To replace the whole endogenous gene with the donor gene, the targeted gene was cleaved around both the start and stop codons by the CRISPR/Cas9 method [52]. To avoid the cleavage of the donor vector, a specific cleavage sequence that doesnot exist within the donor DNA must be selected in the endogenous gene. Therefore, the targeted sequence for CRISPR/- Cas9-mediated cleavage was designed within introns around both the start and stop codons of the gene. In our case, the cDNA of the donor gene contains FLAG and HA tag sequences at the amino (N)- and carboxyl (C)-terminal ends, respectively. Therefore, the DNA sequences around the start and stop codons are different and allow discrimination between the endogenous gene and the donor gene. By selecting the sequence containing the start or stop codon as the CRISPR/Cas9-targeted region, the endogenous gene can be cleaved selectively. For CRISPR/Cas9-mediated cleavage, a 5'-NGG-3′ sequence named the proto-spacer adjacent motif (PAM) is required on the 3′ end of the target sequence. In our case, PAM sequences were found in the vicinities of the start and stop codons of the target gene. Therefore, we could design the CRISPR RNAs to specifically guide Cas9 to the vicinity of the start or stop codon of the targeted endogenous gene. We used the commercially available GeneArt CRISPR Nuclease Vector from Life Technologies for the production of the CRISPR RNA and the Cas9 protein in cells. To generate the knock-in cells, the donor DNA plasmid was transfected into the cells with the two plasmids expressing the CRISPR RNAs targeting the vicinities of the start codon and the stop codon of the targeted gene. The transfected cells were initially cultured in the absence of selectable antibiotics for a few days and subsequently cultured in the presence of appropriate concentrations of the antibiotics for the selection of the knock-in cells. Most of the cells died after the selection. However, the antibiotic-resistant cells were observed after a long cultivation in the same culture dish. The antibiotic-resistant clones were isolated, the genomic DNA from each clone was purified, and the desired knock-in cells were subsequently verified by PCR and sequencing analyses. Thus, we obtained the desired knock-in cells by our method. However, we think that there is still room for improvements of our method.-

In our method, we used circular plasmid DNA as the donor DNA (**Figure 3**). However, the knock-in efficiency is reportedly enhanced when the circular DNA is linearized [53]. Therefore, our strategy might be improved by using linearized donor DNA.-According to the report, the knock-in efficiency of the donor DNA fragment is decreased when nonhomologous terminal DNA regions are present adjacent to the two homology arms [53]. The inhibitory effect of the nonhomologous DNA regions is increased in accordance with the length. Therefore, the most suitable donor DNA is a linearized DNA fragment containing homology arms at both terminal ends, without nonhomologous regions at the ends. When restriction enzyme sites are introduced at the terminal ends of the homology arms of our donor DNA plasmid, this linearized donor DNA can be produced by restriction enzyme cleavage. This modification of our strategy will improve the knock-in efficiency.-

The DSB repair pathway choice is an important consideration to improve the knock-in efficiency. Among the several DSB repair pathways, the HR pathway choice is enhanced when- the NHEJ pathway does not work [64]. Therefore, in order to increase the knock-in efficiency- by HR, the inhibition of the NHEJ pathway was attempted in CRISPR/Cas9-mediated genome- editing [65–68]. A chemical compound, SCR7, inhibits DNA ligase IV, which is an essential- protein for NHEJ [69]. The CRISPR/Cas9-mediated homology-directed genome editing was- enhanced by treating the cells with SCR7 [65, 66, 68]. In addition, a high throughput chemical- screen identified small molecules that modulate CRISPR/Cas9-mediated genome editing [70].- The HR-mediated knock-in efficiency was improved by two chemical compounds, L755507 and- BrefeldinA, which are a β3-adrenergic receptor agonist and an inhibitor of protein transport from the ER to the Golgi apparatus, respectively [70]. In contrast, the HR-mediated knock-in- efficiency was decreased by the chemical compounds azidothymidine (AZT) and Trifluridine- (TFT), which are anti-HIV and anti-herpes virus drugs, respectively [70]. In contrast to their- effects on the HR-mediated knock-in, L755507 inhibits the NHEJ-mediated knock-out, whereas- AZT enhanced it [70]. Therefore, in addition to their known activities, these chemical compounds could modulate the DSB repair pathway choice. The effectiveness of L755507 for the- HR-mediated knock-in was also demonstrated in another study [68]. Therefore, the knock-in- strategy could be further improved in combination with the usage of these chemical compounds.-
