Fig. 1: Circularization of ssDNA increases the overall efficiency of TALEN-mediated knock-in in HSPCs.

a HSPCs editing protocol using an mRNA-encoded TALEN targeting the B2M locus and LssDNA or CssDNA as DNA donor templates to insert a tag (0.6 kb) or a reported gene (2.2 kb) via non-disruptive and disruptive insertions, respectively (LHA and RHA stand for right and left homology arm, respectively). Created in BioRender. Valton, J. (2025) https://BioRender.com/7qzeb34. mRNAs encoding a viability enhancer and a HDR enhancer (Via-Enh01 and HDR-Enh01, respectively) were also incorporated in the first transfection. The timing is indicated in days (D0-D7). Edited HSPCs obtained 7 days post-thawing (D7) were characterized by flow cytometry to assess the level of phenotypic knock-in (KI) of DNA donor templates and phenotypic knock-out (KO) of B2M as well as their viability. Their differentiation capacity into erythroid and myeloid progenitors was also assessed by colony forming unit (CFU) assay. b Flow cytometry results illustrating the frequency of cells harboring KI events, the ratio KI/KO, the viability and plating efficiency of HSPCs either untreated, electroporated (Mock EP), edited with TALEN only (TALEN), or edited with TALEN and LssDNA or CssDNA donor templates (LssDNA or CssDNA, respectively). Top and bottom panels illustrate results obtained with 0.6 kb and 2.2 kb DNA donor templates, respectively. On each box plot, the central mark indicates the median, the bottom and top edges of the box indicate the interquartile range (IQR), and the whiskers represent the maximum and minimum data point. Each dot represents data obtained from one HSPC donor (numbers of independent donor, i.e., biological replicate, are indicated underneath of each plot). Mann–Whitney two-tailed non-parametric unpaired test with a confidence interval of 95%. P-values are indicated. Source data are provided as a Source data file.