Figure 5.

Simultaneous insertion of paired LoxP sites into the same allele by Bi-PE. (A) Schematic diagram showing the design and the putative editing process of Bi-PE mediated insertion of paired LoxP sites. (B) PCR analysis of the targeted insertions of LoxP sites. Upper and middle panels were schematic diagrams showing the designs of the PCR primers and the putative outcomes of single or double LoxP insertions. Lower panels were agarose gels analysis of the PCR products with or without EcoRV digestion. (C and D) Quantifying the efficiency and purity of the targeted insertion of double LoxP sites through single colony analysis. PCR products from (B) were cloned into pESI-blunt, and the resulting single colonies were analyzed by PCR and Sanger sequencing. Colonies containing perfect double-LoxP insertions were recognized as accurate editing and the ones containing double-LoxP insertions but harboring indels were inaccurate. Detailed information of the indels were shown in Supplementary Figures S12 and S13.