Figure 4. Knockdown and knockout of NHEJ-related genes increased the HR activity in silkworm cells.

(a) A schematic representation of plasmids for HR reporter system. This system was constructed before¹⁸. (b) BmN4-SID1 cells were treated with dsRNAs against NHEJ-related genes for 3 days, followed by co-transfection with pSK8Luc5′3′DR and pPBO-I-SceI-FW-r. Another 3 days later, cells in each well were separated into two parts for Luc assay and western blot, respectively. Cells treated without dsRNA (Mock) was used as a negative control. (c) Mutant cells were prepared in 24-well plates at the density of 1 × 10⁵ cells/well for 16 h, followed by co-transfection with the reporter plasmids. Luc assay and western blot were performed after 3 days later. The cell line named N4-SID1-Cas9, which transgenically expressed Cas9 protein in BmN4-SID1 cells, was used as a negative control. In B and C, the mean fold (the controls were set as 1 fold indicated by dotted lines) was derived from values of luciferase assay normalized by the total protein concentrations of cells in each treatment. Differences between the means were evaluated with a two-tailed Student t-test. Significant differences are as follows: **P < 0.01, ***P < 0.001. The western blot data was a representative from repeated three independent experiments.