Fig. 3

Targeted genome editing and transcriptional activation of porcine endogenous genes using the DIC system. a Target genes and sgRNA sequences used to detect editing efficiency. b Efficiency of genome editing for target genes shown in (a) after transfecting corresponding sgRNA into DIC PFFs. c Schematic of the chromosome engineering events in the porcine ALK and EML4 loci after simultaneously transfecting ALK-sgRNA and EML4-sgRNA into DIC PFFs. d PCR analysis and Sanger sequencing of chromosomal inversion and large fragment deletion between ALK and EML4 loci. e RT-PCR analysis and Sanger sequencing of EML4-ALK fusion transcripts resulting from chromosomal inversion between ALK and EML4 loci. In d and e, “+1” group refers to the simultaneous transfection of two plasmids containing ALK-sgRNA and EML4-sgRNA, respectively; “+2” group refers to transfection of a single plasmid containing pre-tRNA-linked ALK- and EML4-sgRNAs. f Schematic of transcriptional activation constructs. MPH, MS2-P65-HSF1 complexes controlled by CMV promoter. dgRNA, nuclease-dead single guide RNA, driven by U6 promoter, harboring MS2-loop to recruit MS2-P65-HSF1 complexes and a 14-bp target sequence, which inactivates SpCas9. dgRNA 1-6, representing six dgRNAs driven by its own U6 promoter in tandem. g Schematic of the targeted transcriptional activation of porcine endogenous genes using the DIC system. h–i Q-PCR results in DIC PFFs transfected with MPH and different dgRNAs, presented as mean ± SEM, to measure the transcript level of CDX2 locus (h) and SOX2 locus (i) normalized to control cells from three independent experiments (n = 3). DIC PFFs transfected with only MPH were used as control cells