Fig. 5

NHEJ-mediated knock-in of BDDF8 at Alb introns 11 or 13 cures hemophilia A. a Schematic of the intron targeting of BDDF8 at the mouse Alb locus. We used ten sgRNAs in this study. The yellow box indicates the Alb sequence on the donor: 3′ end of intron 13 (46 bp) and 5′ end of exon 14 before the stop codon (39 bp), which serves as a splice acceptor. After cut at an Alb intron, the linearized donor will integrate via the NHEJ repair pathway. Right panels show the predicted fusion mRNA and protein products. b Assessment of sgRNA cleavage efficiency by Illumina sequencing 1 week after injection of Cas9-sgRNA. An unpaired t test with Welch’s correction was used for statistical analysis; ***P < 0.001. c Differential F8 activity after knock-in of BDDF8 donor at different loci of the Alb gene. sgRNAs targeting intron 1 and intron 12 serve as negative controls. d Analysis of Alb-BDDF8 fusion transcripts by RT-PCR. e DNA sequencing data confirmed the correct splicing of different Alb exons or skipping an Alb exon or inclusion of an exon from the plasmid backbone