Enhancing the antibacterial activities of sow milk via site-specific knock-in of a lactoferrin gene in pigs using CRISPR/Cas9 technology

Abstract

Colostrum quality is a vital factor in mortality and growth performance for piglets. Lactoferrin is an immuno-active milk protein that contributes to the formation of a protective layer above intestinal mucosa, possesses the antibacterial and antiviral activities that are favorable for piglet development. However, there is a notable reduction in lactoferrin in sow milk during lactation after the first few days, which causes many piglets to fail to ingest enough colostrum thereby leading to an increase in piglet mortality. In this study, we successfully constructed genome-edited Large-White pigs with marker-free site-specific knock-in of lactoferrin gene in the 3'-end of Casein alpha-s1 via CRISPR/Cas9 mediated homologous recombination. Thus, the lactoferrin protein can be expressed in the mammary gland in the control of Casein alpha-s1 promoter. As expected, the lactoferrin protein in genetically modified pigs sustained high expression in both colostrum and milk when compared with wild-type pigs. Moreover, the bacterial plate assay indicated that the milk from genetically modified pigs showed bacteriostatic effects when compared with control pigs. Taken together, our study demonstrated that the milk from genetically modified pigs had antibacterial activity which may reduce the costs of veterinary drug and improve the surviving rate of piglets, which is promising for pig breeding.

Keywords: CRISPR/Cas9; Colostrum; Homologous recombination; Lactoferrin; Pig.

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

Fig. 2

Generation of site-specific lactoferrin knock-in pigs using CRISPR/Cas9 technology. a Detection of site-specific insertion of selected clones using PCR genotyping. Lanes 1–12 represent 12 different clones, lanes 2, 3, 7, 9, 10 represent clone 25#, 27#, 68#, 75# and 81#, respectively; lane N represents the negative control wild-type cell line. b A genetically modified piglet at day 3 postpartum. c Detection of site-specific insertion of donor DNA in the genetically modified pigs using a PCR genotyping assay. Lanes 1–7 represent 7 individuals, and lane WT represents one negative control wild-type pig. WT, wild-type. d Nucleotide sequence analysis of junctions between endogenous and exogenous DNA corresponding to homologous recombination (HR) events. P5/P6 primers were used to amplify the specific region for the left- and right-hand junctions. Primary nucleotide sequence data corresponding to transition regions between the homologous arms of the targeting vector and the internal transgene DNA. LF, lactoferrin

Fig. 3

Detection of the antibacterial activity of milk from pLF-KI pigs in vitro. a Raw components of genetically modified colostrum compared with wild-type colostrum. b Detection of the expression of LF in colostrum stage and milk stage in pLF-KI pigs by qRT-PCR assay. c Western blot analysis for the expression and accumulation of LF protein between pLF-KI pigs and wild-type pigs. KD, Kilodalton. d Bacterial plate assay for Escherichia coli (E. coli) survival in response to colostrum treatment. Depletion zones developed on a lawn of E. coli 12 h after sample application: (1) 10 μL milk from genetically modified pig, (2) 10 μL milk from wild-type pig, (3) 10 μL sterile water and (4) 10 μL antibiotic. e Plate dilution colony counting assay to test the antibacterial activity of colostrum. pLF-KI, porcine lactoferrin knock-in; WT, wild-type; Anti, Antibiotic; CFU, colony-forming unit; Blank: sterile water; the cycles in orange indicate the transparent zones. ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001; P values were determined by two-sided Student’s t test