Efficient RNA-guided base editing for disease modeling in pigs

a Schematic of the PB-BE3 vector. CMV cytomegalovirus promoter, rAPOBEC1 rat cytidine deaminase apolipoprotein B editing complex 1, Cas9n (D10A) Cas9 nickase with D10A mutation, UGI Uracil DNA glycosylase inhibitor, SV40 SV40 promoter, neo neomycin resistant gene, hU6 human U6 promoter, sgRNA single guide RNA, PB3 and PB5 terminals of the piggyBac transposon. b, c The target sequence at the TWIST2 and TYR loci. Missense (E75K) and nonsense (Q68Stop) mutations were introduced by BE3 in TWIST2 and TYR gene, respectively. The base editing window of BE3 is approximately 5？nt, typically -17 to -13 upstream of the PAM sequence. The asterisk represents the target site. The PAM sequence is shown in cyan. The sgRNA target sequence is underlined. The nucleotides and amino acids substituted by BE3-mediated base editing are shown in red and magenta. d, e Representative sequencing chromatograms of porcine fetal fibroblast clones at the TWIST2 and TYR loci. The relevant codons are underlined in red. f Compared with wild-type newborn piglet (top panel), TWIST2 mutant piglets (middle panel) exhibit severe deformities such as macrostomia, microtia, and absent eyelids. Representative genotypes of TWIST2 mutant piglets are illustrated at the bottom panel. g The white piglet is TYR mutant exhibiting a typical albinism phenotype, compared with the black one on the left with a heterozygous mutation. Representative genotypes of TYR mutant piglets are illustrated at the botto