Fig. 2

NHEJ and HDR mediated BDDF8 knock-in at Alb stop codon. a Schematic of genome editing at the Alb stop codon. Knock-in of promoterless BDDF8 expression cassette at Alb through NHEJ or HDR was achieved by Cas9-sgAlb mediated simultaneous cleavage of the genome and the double-cut donor pD-BDDF8-sg. The pD-sg template carries 600-bp homology arms. Knock-in by NHEJ or HDR mechanism can be differentiated by the size of the PCR products. HDR = 800 bp and NHEJ = 1400 bp. The left homology arm spans from the middle of exon 13 to the sgAlb target site. The right homology arm spans intronic sequence 3′ of the sgAlb target site. After integration and transcription by the endogenous Alb promoter/enhancer, two proteins (Alb and BDDF8) are produced as the result of E2A-mediated ribosomal skipping. polyA, polyadenylation site; WPRE, Woodchuck hepatitis virus (WHP) posttranscriptional regulatory element. b Editing with the double-cut BDDF8 donor restores F8 activity in hemophilia A (n = 5). Treatments without one or two editing component (n = 4 for each) serve as negative controls. An unpaired t test with Welch’s correction was used for statistical analysis; ***P < 0.001. c PCR analysis showing gene targeting mediated by both HDR and NHEJ. Liver samples were harvested 1 week after hydrodynamic injection of Cas9-sgAlb and the donor. We analyzed both the left and the right junctions by PCR. The locations of primers are indicated in a. PCR products were resolved by 2% agarose gel. gDNA from untreated mice (WT) serves as a negative control. d The identity of the NHEJ and HDR PCR products was confirmed by sequencing. Shown is the Sanger sequencing data of the left junction. e PCR analysis showing a successful fusion of Alb and BDDF8 1 week after hydrodynamic injection of Cas9-sgAlb and donor vectors. f DNA sequencing data confirm the correct splicing of exon 13 and exon 14, and the fusion of the E2A-BDDF8 cassette