Fig. 1

Construction of CRISPR plasmids and activity assessment. a Schematic representation of the design of the CSN1S1-targeting vector. Exons of CSN1S1 and homologous arms are shown as gray boxes, and the purple triangle in Exon 17 represents the sgRNA targeting site. The pLF gene flanked by P2A and polyA in green is located in the middle of the targeting vector. Three pairs of primers were designed for genotyping. P1/P2 and P3/P4 are designed for genotyping the 5′ and 3′ junctions in pLF-KI colonies, respectively. P5/P6 are designed for detecting the donor vector region within the genetically modified pigs. LF, lactoferrin; HR, Homologous Recombination; sgRNA, single-guide RNA. b Assessment of the activity of 18- and 20-nt sgRNAs targeted to the same genomic loci via the T7ENI cleavage assay in vitro. The upper sequences indicate the 18- and 20-nt sgRNAs, respectively, with the protospacer adjacent motif (PAM) sequence underlined. NC, negative control; M, DL2000 Marker. c Verification of activity of different length sgRNAs by Sanger sequencing. Characters in purple indicate the sgRNA sequences; The PAM sequences are underlined; Δ, deletion; +, insertion; WT, wild-type; No., Numbers of Sanger sequencing results