Fig. 2: Addition of RAD51-preferred ODNs increases ssDNA donor-mediated HDR efficiency at the BFP locus.
From: Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor
a Schematic representation for gene editing in the BFP reporter cell lines. Single-copy-integrated BFP cell lines were electroporated with Cas9 RNP together with modular or canonical ssDNA donors. Then fluorescence conversion percentage was measured using flow cytometry. b, c HDR efficiencies of ssDNA donors connected with RAD51- or Ku80-preferred ODNs in HEK 293T-BFP cells (b) and K562-BFP cells (c). d HDR efficiencies of ssDNA donors at different concentration gradients in HEK 293T-BFP cells. e The effect of the incorporation position on the HDR improvement of HDR-boosting modules. f HDR efficiencies of ssDNA donors incorporated with the indicated mutant SSO14 modules. g Schematic representation for gene editing with ssDNA donors and independent HDR-boosting modules. h HDR efficiencies of ssDNA donors in HEK 293T-BFP cells electroporated with CRISPR-Cas9 RNP, canonical ssDNA donor, and independent HDR-boosting module. i Evaluation of the binding activity of RAD51 with ssDNA donors using the ODIP assay. Donor mix, an equal parts mix of the three ssDNA donors mentioned in the image. j Western blot analysis of the knockdown efficiencies of three siRNAs targeting RAD51 in HEK 293T cells 48 h post-transfection. siRAD51-mix, an equal parts mix of the three siRNAs of RAD51 mentioned in the image. k, l Effects of RAD51 knockdown on HDR efficiencies of ssDNA donors at the BFP site in HEK 293T-BFP cells (k) and endogenous FANCF site in HEK 293T cells (l). For all HDR efficiency-assessing experiments unless otherwise specified, 18 pmol Cas9 nuclease, 22 pmol gRNA and 6 pmol ssDNA donors corresponded to 2 × 105 cells. HDR efficiency was measured three days after electroporation. Data are representative of 3 independent experiments (i, j). Values and error bars reflect mean ± SD of n = 3 (b, d–f, h, k) independent electroporation replicates. Values reflect n = 2 (c, l) independent electroporation replicates. The sequences of all gRNAs, ssDNA donors and siRNAs used are shown in Supplementary Data 1, 2, and 6. Source data are provided as a Source Data file. Figure (a, g) Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
