Fig. 4

BDDF8 insertion at the Alb stop codon is predominantly through NHEJ. a Schematic of genome editing at the Alb stop codon using double-cut BDDF8 donors with different lengths of homology arms. The pD-sg templates carry the different length of homology arms (HA600-600, HA190-130, HA190-0, HA85-130, HA85-0), flanked by Cas9-sgDocut recognition sequences. NHEJ- or HDR-mediated knock-in can be differentiated by the size of the PCR product using primers F1 and R1. Successful integration leads to the transcription of an Alb-BDDF8 fusion gene, which translates to two proteins: Alb and BDDF8. b High-level F8 activity 1 week after injection of Cas9-sgAlb and double-cut pD-BDDF8-sg donors with different length of homology arms (n = 2–5 for each group). Mice treated without donor only (n = 2) serves as a negative control. An unpaired t test with Welch’s correction was used for statistical analysis; ***P < 0.001. c PCR analysis showing successful gene targeting by both HDR and NHEJ. PCR analysis of the left junction in edited mice. The locations of F1 and R1 primers are shown in a. PCR products were resolved on a 2% agarose gel. Untreated mice (WT) showed no evidence of targeting. d Quantification of NHEJ and HDR editing at the left junction using ddPCR. Liver gDNA was extracted 1 week after editing using donor pD-BDDF8(HA85-130). We used probes targeting both the junction (NHEJ) and HA85 (NHEJ+HDR) in ddPCR. e Amplification of the fusion transcript of Alb and BDDF8 by RT-PCR. f Sanger sequencing data show correct splicing of exon 13 and exon 14 and the exon 14-E2A junction