Journal: iScience

Article Title: Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha

doi: 10.1016/j.isci.2021.102168

Figure Lengend Snippet: A competitive binding to DSBs leads to the change in HR/NHEJ ratio (A) HR colonies and NHEJ colonies in strains of wild-type (WT), KU80 down-regulation (Ku80-dw under repression of methionine), and overexpression of HR-related proteins (y34), which were calculated by multiplication of total colonies and HR rates (blue square). (B) Schematic illustration of a competitive mechanism of HR (RPA) and NHEJ (Ku80). For controlling DSBs formation well, an inducible gRNA plasmid targeting LSC2 gene was constructed by using a methanol-induced promoter P DAS1 . The relative abundance of RPA-GFP and Ku80-RFP was detected by fluorescence intensity. (C) Fluorescence intensities of GFP (B) and RFP (C) were measured at 24 h in strains WT, Ku80-dw, and y34. In particular, 1.7 mM methionine was added to repress the Ku80 expression in strain Ku80-dw (Ku80-dw + Met). Cells were cultivated in Delft basic salt media containing 10 g/L methanol (Induction), or 20 g/L glucose (No induction), at 37°C, 220 rpm. (D) The relative abundance of RPA and Ku80, which was calculated by the ratio of GFP fluorescence intensities and RFP fluorescence intensities, was highly consistent with the corresponding HR rates. Data are presented as means of three biologically independent samples with displayed data points. Red asterisks indicate statistical significance as determined using paired t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Article Snippet: gRNA plasmids (pHpgRNA13 and pHpgRNA50) generated in this study have been deposited to Addgene (Yongjin Zhou, 78587).

Techniques: Binding Assay, Over Expression, Plasmid Preparation, Construct, Fluorescence, Expressing