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In order to analyse pseudo attP site-specific excision by Int mutants, pseudo attP sites were

derived from favoured genomic PhiC31 integrase target sites within the mammalian genome and inserted into the substrate plasmid. HEK 293 cells were co-transfected with integrase encoding plasmids and the substrate plasmid at a low Int dose pLuc:Int = 1:1 and at a high Int

dose pLucCR:Int = 1:5. First, the pseudoattP site derived from chromosomal location 2q11.2

Comparing the results from the two experiments with different plasmid ratios showed that the relative fold change is higher in 9 of 21 mutants when a high amount of Int plasmid was transfected (magenta bars in Figure 4.27). Only mutants E382A, E383A, and K450A showed about 33 % higher excision activities at low dose of Int plasmid (pLucCR: Int = 1:1; blue bars) compared to a plasmid ratio pLucCR:Int = 1:5 (magenta bars).

Twelve mutants, K367A, E371A, R380A, R386A, R390A, R394A, D398A, D417A, R432A, R429A, R448A and D470A, showed no significant difference in excision activity between a high and a low dose of Int plasmid. In both experiments, a few mutants showed improved excision compared to wt Int. At a plasmid ratio of 1:1 five Int mutants showed higher excision activity compared to wt Int (blue bars > 100 %). At a plasmid ratio of pLucCR:Int = 1:5, nine mutants showed higher excision activity compared to wt Int (magenta bars > 100 %). However, five of them showed only minor improvements up to 1.25-fold. The negative control mInt dropped only about 60 % compared to wt Int in both ratios.

Figure 4.27. Excision activity of integrase mutants at pseudoattP site 2q.11.2 (abbreviated as hs2). Two assays were performed with a 1: 1 ratio (blue bars) and a 1: 5 ratio (magenta bars) of transfected plasmids pLucCR: Int plasmid. A total of 2000 ng plasmid DNA was transfected per well. Relative light units (RLU) of wt Int were set to 100 %. Experiments were carried out in triplicate.

Next, the pseudoattP site derived from 12q.22 was analysed for recombination (Figure 4.28).

When Int plasmid was transfected fivefold in excess compared to substrate plasmid pLucCR, improved excision activity in 14 of 21 Int mutants was observed. Several Int mutants showed

lower excision activity than wt Int in the context of pseudo attP site 12q.22. Only mutant

dose. The excision activity of mutant R429A increased 2.7-fold when increasing the integrase plasmid dose compared to substrate plasmid. Sixteen mutants showed lower excision activity between 20 % and 80 % compared to wt Int at both plasmid ratios. The excision activity of mInt dropped to 30 % and 45 %.

Figure 4.28. Excision activity of integrase mutants at pseudo attP site 12q.22 (hs12). Two assays were performed with a 1:1 ratio (blue bars) and a 1:5 ratio (magenta bars) of transfected plasmids pLucCR:Int plasmid. A total of 2000 ng plasmid DNA was transfected per well. Relative light units (RLU) of wt Int were set to 100 %. Experiments were carried out in triplicate.

Furthermore, the excision based assay was performed with substrate plasmid pLucCR

containing pseudo attP site 19q.13.31 (Figure 4.29). The excision activity of integrase

mutants at recombination target sites attB _× attP´ (19q13.31) confirmed that slightly higher

excision activities were obtained when more Int plasmid was transfected. Only two mutants, K367A and K450A, showed a decrease of 30-40 % after enhancing Int plasmid dose. The mutant E371A obtained the highest excision efficiency with almost twofold increase at both plasmid ratios. Twelve mutants showed excision activities lower than wt Int-mediated excision. At a 1:1 ratio, the mutants E371A and K450A showed twofold and 1.4-fold higher excision activity than wt integrase. At high integrase plasmid dose, the mutants E371A, E382A, and E406A obtained increased excision activities between about 1.5- and twofold, compared to wt Int. The decrease of mInt compared to wt Int to 30 % and below at both low and high integrase plasmid dose proved the functionality of the assay.

Figure 4.29. Excision activity of integrase mutants at pseudoattP site 19q13.31 (hs19). Two assays were performed with a 1:1 ratio (blue bars) and a 1:5 ratio (magenta bars) of transfected plasmids pLucCR: Int plasmid. A total of 2000 ng plasmid DNA was transfected per well. Relative light units (RLU) of wt Int were set to 100 %. Experiments were performed in triplicate.

Taken together, analysing the site-specific excision activity of integrase mutants in the context

of different pairs of recombination recognition sites, wt attB _× wt attP, wt attB _× attP´ (at

chromosomal position 2q11.2), wt attB_×attP´ (12q.22), and wt attB_×attP´ (19q.13.31) in an

intramolecular plasmid-based recombination assay showed high variations. Differences in recombination activity were not only observed among mutants recombining the same pair of

recognition sites but also within single mutants at different pseudo attP sites. Individual

mutants showed increased excision activity when the introduced pseudo attP sites were

targeted. However, whether these mutants might integrate into the tested pseudo attP sites in

context of genomic DNA as well, is highly influenced by variables like accessibility and potential preference for competing integration sites.

A summary showing relative recombination activities of integration and excision assays obtained from colony-forming assays and the GFP and luciferase reporter systems is presented in Table 4.2 below. Improved activities compared to wt integrase are presented in bold. The double mutants and D470A showed 1.5-fold to threefold improved integration efficiency in HeLa and HCT cells, and the mutants E371A, E382A, E383A, E406 and K450A showed between 1.3 and 2.2-fold excision activities in luciferase assays at different recognition sites.

Table 4.2. Overview of PhiC31 integrase-mediated recombination activities in colony-forming assay, FACS and luciferase assay carried out in vitro. The recombination activity for each mutant is given in percentage compared to wt integrase (100 %). Plasmid ratios and plasmids are described.

Assay Colony-forming assay FACS Luciferase assay

cell line HeLa HCT Huh7 293 Hep1A #28 293

figure 4.7 4.8 4.10 4.11 4.12 4.13 4.14 4.15 4.21 4.23 4.24 4.27 4.28 4.29 plasmid names substrate plasmid p7: Integrase plasmid substrate plasmid pLucCR : integrase plasmid plasmid ratio 1:0.5 1: 1 1:0.5 1:20 1:1 1:20 1:1 1:10 0.9:1 1:5 1:1 1:5 1:1 1:5 1:1 1:5

attB/ attB sites all targeted attP´sites within particular cell lines attB×attP attB×attP´ attB ×attP´ attB ×attP´

position of attP native (2q11.2) (12q.22) (19q13.31)

wt integrase 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 D366A 1.22 0.63 0.97 0.58 0.94 1.37 0.82 0.87 1.00 1.06 K367A 0.10 0.07 0.05 0.43 0.99 1.21 0.39 0.74 0.78 0.52 E371A 0.90 0.62 1.94 1.63 1.44 1.58 1.06 0.93 2.04 1.89 R380A 0.16 0.36 0.31 0.33 0.60 0.74 0.40 0.53 0.41 0.28 E382A 1.36 1.36 1.01 0.70 1.02 1.80 1.23 1.34 1.49 0.77 1.44 E383A 1.28 0.83 1.87 1.32 2.16 1.25 0.95 1.16 0.77 1.10 K386A 0.61 0.72 1.34 1.50 0.56 0.76 0.35 0.45 0.48 0.63 R390A 0.60 0.64 1.15 0.79 0.91 0.89 0.91 0.45 0.64 0.58 R393A 0.05 0.35 R394A 0.13 0.85 0.89 0.69 0.59 0.83 0.36 0.42 0.41 0.55 D398A 0.76 1.07 1.02 0.65 0.73 0.83 0.46 0.61 0.67 0.76 E406A 0.86 1.03 1.93 0.83 1.28 1.73 1.02 0.71 0.58 1.56 D417A 0.03 0.04 0.04 1.08 0.35 0.47 0.20 0.34 0.16 0.22 R423A 0.81 0.88 1.75 0.90 0.74 0.74 0.63 0.53 0.76 0.83 R429A 0.60 1.11 0.77 0.85 1.03 1.06 0.62 1.69 0.53 0.91 E432A 0.09 0.53 0.12 0.78 0.03 1.78 0.01 0.71 0.00 0.73 E435A 0.77 1.05 1.19 0.85 0.81 1.23 0.60 0.82 0.57 1.05 R446A 1.04 0.95 1.01 0.91 0.69 0.86 0.53 0.51 0.56 0.63 K450A 0.04 0.68 2.20 0.40 1.57 0.98 0.72 0.74 1.40 1.04 K457A 1.20 1.66 1.95 0.7 1.17 1.3 1.02 0.89 0.87 0.69 1.12 0.72 0.78 1.08 1.05 R461A 1.14 0.94 0.20 0.73 0.15 1.01 0.16 0.92 0.09 1.02 D470A 1.70 1.47 1.45 0.42 1.05 1.09 0.60 0.84 0.38 0.86 0.89 0.74 0.60 0.65 1.02 E382A+D470A 1.70 3.3 1.27 2.33 1.44 1.3 K457A+D470A 1.10 3.0 1.43 1.46 1.19 0.63

Based on these results, a few mutants were investigated for their detailed integration specificity in order to determine the specific location of integrase-mediated insertion within the genome of HCT cells.