Generation of Anti-Mastitis Gene-Edited Dairy Goats with Enhancing Lysozyme Expression by Inflammatory Regulatory Sequence using ISDra2-TnpB System

Abstract

Gene-editing technology has become a transformative tool for the precise manipulation of biological genomes and holds great significance in the field of animal disease-resistant breeding. Mastitis, a prevalent disease in animal husbandry, imposes a substantial economic burden on the global dairy industry. In this study, a regulatory sequence gene editing breeding strategy for the successful creation of a gene-edited dairy (GED) goats with enhanced mastitis resistance using the ISDra2-TnpB system and dairy goats as the model animal is proposed. This included the targeted integration of an innate inflammatory regulatory sequence (IRS) into the promoter region of the lysozyme (LYZ) gene. Upon Escherichia Coli (E. coli) mammary gland infection, GED goats exhibited increased LYZ expression, showing robust anti-mastitis capabilities, mitigating PANoptosis activation, and alleviating blood-milk-barrier (BMB) damage. Notably, LYZ is highly expressed only in E. coli infection. This study marks the advent of anti-mastitis gene-edited animals with exogenous-free gene expression and demonstrates the feasibility of the gene-editing strategy proposed in this study. In addition, it provides a novel gene-editing blueprint for developing disease-resistant strains, focusing on disease specificity and biosafety while providing a research basis for the widespread application of the ISDra2-TnpB system.

Keywords: ISDra2‐TnpB system; gene‐editing breeding strategy; goats; mastitis.

Figure 1

The anti‐inflammatory effects of dairy goat LYZ were analyzed by RNA‐seq. The cells were divided into 3 groups for treatment: group A (n = 3 per group) was the control group, group B (n = 3 per group) was treated with 5 µg mL⁻¹ LPS for 12 h and group C (n = 3 per group) was treated with 5 µg mL⁻¹ LPS for 12 h, and then 10 nM dairy goat LYZ recombinant protein was treated for 12 h. a) Bubble map obtained by GO analysis of RNA‐seq data from group B and C, with enrichment factors represented by horizontal coordinates. Bubble color and size correspond to the ‐log₁₀(p_value). b) Bar graph obtained by GO analysis of RNA‐seq data from groups B and C, where the horizontal coordinate is the ‐log₁₀(p_value). c) Bubble map obtained by KEGG analysis of RNA‐seq data from group B and C, where the horizontal coordinate is the enrichment factor. Bubble color and size correspond to the ‐log₁₀(p_value). d) Bar graphs obtained by KEGG analysis of RNA‐seq data from groups B and C. Bar color correspond to the p.adjust. e) Heatmap analysis of RNA‐seq data from group A, B and C. f) Analysis of the apoptosis‐NF‐κB‐necrotosis interaction pathway was performed on the RNA‐seq data of group B and C. Bubble color and size correspond to the log₂(fold change).

Figure 2

Screening of IRS. a,b) The pGL4.10‐1/−2000 vector was transfected into HEK293T cell and treated with LPS of different concentrations for 12 and 24 h to detect promoter activity. c) The pGL4.10‐1/−2000, pGL4.10‐1/−1500, pGL4.10‐1/−1000 and pGL4.10‐1/−500 vector were transfected into HEK293T cells, treated with 5 µg mL⁻¹ LPS for 12 h to detect promoter activity. d,e) The pGL4.10‐IRS‐LYZ vector was transfected into primary GMEC and HEK293T cells, treated with 5 µg mL⁻¹ LPS for 12 h to detect promoter activity. Values are expressed as mean ± SEM (n = 3 per group) by one‐way ANOVA. *: indicates significant difference (P < 0.05), **: indicates that the difference is highly significant (P < 0.01) and ns: indicates no significant difference (P > 0.05).

Figure 3

Analysis of anti‐inflammatory effects of gene‐edited GMEC. a) Gene editing schematic. b) Analysis of cleavage activity of six sgRNAs (n = 3 per group). c) Gene‐edited GMEC status before and after transfection with pCNDA3‐Cre vector. Scale bar: 100 µm. d,e) Sanger sequencing results of positive monoclones with green fluorescent screening markers and without screening markers. f) Primary GMEC and gene‐edited GMEC were treated with LPS for 12 h, and then the culture medium was replaced for 12 h to detect the protein expression level of TLR4, MyD88, p‐IKBα, IKBα, p‐NF‐κB‐p65 and NF‐κB‐p65 (n = 3 per group). g) Analysis of LYZ protein expression. Values are expressed as mean ± SEM (n = 3 per group) by one‐way ANOVA. *: indicates significant difference (P < 0.05), **: indicates that the difference is highly significant (P < 0.01) and ns: indicates no significant difference (P > 0.05).

Figure 4

Production of LYZ GED goats by SCNT. a) Schematic diagram of SCNT. b) Gene‐edited GFFs status before and after transfection with pCNDA3‐Cre vector. Scale bar: 100 µm. c) Developmental status of embryos after SCNT. Scale bar: 100 µm. d,e) Sanger sequencing results of positive monoclones with green fluorescent screening markers and without screening markers. f) Production data of GED goats. g) Three GED goats. h) Identification of GED goats.

Figure 5

GED goats with high LYZ expression reduce the severity of mastitis and alleviate BMB damage. a,b) The status of GED goats and WTD goats after infection with E.coli for 7 d. c) SCC in milk during E. coli infection (n = 3 per group). d) Expression level of LYZ protein in GED goats milk 15 d before infection (n = 3 per group). e) Expression level of LYZ protein in WTD goats and GED goats milk after infection (n = 3 per group). f,g) HE staining of mammary gland tissue of WTD goats during lactation after 7 d of sterile PBS and E.coli injection, respectively. Scale bar: 100 µm. h) HE staining of mammary gland tissue of GED goats during lactation after 7 d of E.coli injection. Scale bar: 100 µm. i,j) ZO‐1staining of mammary tissue during lactation of normal WTD goats during lactation after 7 d of sterile PBS and E.coli injection, respectively. Scale bar: 200 µm. k) ZO‐1 staining of mammary tissue during lactation of GED goats during lactation after 7 d of E.coli injection. Scale bar: 200 µm. l–n) Analysis of content of IL‐6, TNFα, and IL‐1β in milk (n = 3 per group). Values are expressed as mean ± SEM (n = 3 per group) by one‐way ANOVA. *: indicates significant difference (P < 0.05), **: indicates that the difference is highly significant (P < 0.01) and ns: indicates no significant difference (P > 0.05).

Figure 6

Gene‐edited GMEC alleviates inflammation and TJ damage by high expression of LYZ. a) Flowchart of GMEC organoid establishment. b) GMEC organoid culture 3, 6, 9 and 12 d status. Scale bar: 100 µm. c) CD49f staining of GMEC organoids was performed using the IF method. Scale bar: 100 µm. d–g) Analysis of LYZ, IL‐6, TNFα, IL‐1β protein expression. h) Primary GMEC and gene‐edited GMEC were treated with 5 and 10 µg mL⁻¹ LPS for 12 h, and then the culture medium was replaced for 12 h to detect the protein expression level of TLR4, MyD88, p‐IKBα, IKBα, p‐NF‐κB‐p65, NF‐κB‐p65, ZO‐1 and Occludin. i) The protein expression of p‐NF‐κB‐p65 in the nucleus was detected by IF. Scale bar: 20 µm. j) The protein expression of p‐NF‐κB p65 in GMEC organoids was detected by IF. Scale bar: 100 µm. Values are expressed as mean ± SEM (n = 3 per group) by one‐way ANOVA. *: indicates significant difference (P < 0.05), **: indicates that the difference is highly significant (P < 0.01) and ns: indicates no significant difference (P > 0.05).

Figure 7

Dairy goat LYZ exerts anti‐inflammatory effects by down‐regulation of HMGB1 expression in gene‐edited GMEC. a) Primary GMEC and gene‐edited GMEC were treated with 5 µg mL⁻¹ LPS for 12 h, and then the culture medium was replaced for 12 h to detect the protein expression level of HMGB1. b) The protein ratio of HMGB1 and β‐actin. c) Primary GMEC was treated with 1 µg mL⁻¹ HMGB1 recombinant protein followed by treatment with 10 nM dairy goat LYZ recombinant protein for 12 h to detect the protein expression levels of p‐NF‐κB‐p65, NF‐κB‐p65, IL‐6, TNFα, and IL‐1β. d) The protein ratio of p‐NF‐κB‐p65 and NF‐κB‐p65. e–g) The protein ratio of IL‐6, TNFα, IL‐1β and β‐actin. h) Gene‐edited GMEC was treated with 1 µg mL⁻¹ HMGB1 recombinant protein, 40 µM Glyeyrrhizin and 5 µg mL⁻¹ LPS to detect the protein expression levels of p‐NF‐κB‐p65, NF‐κB‐p65, IL‐6, TNFα, and IL‐1β. i) The protein ratio of p‐NF‐κB‐p65 and NF‐κB‐p65. j–l) The protein ratio of IL‐6, TNFα, IL‐1β and β‐actin. m) Analysis of LYZ protein expression. Values are expressed as mean ± SEM (n = 3 per group) by one‐way ANOVA. *: indicates significant difference (P < 0.05), **: indicates that the difference is highly significant (P < 0.01) and ns: indicates no significant difference (P > 0.05).

Figure 8

Gene‐edited GMEC inhibits PANoptosis activation by high expression of LYZ. a) Primary GMEC and gene‐edited GMEC were treated with 5 µg mL⁻¹ LPS and 10 µg mL⁻¹ LPS for 12 h, and then the culture medium was replaced for 12 h to detect the protein expression levels of GSDMD, NLRP3, Caspase 1, Cle‐Caspase 1, ASC, Caspase 8, Cle‐Caspase 8, Bcl2, Caspase 3, p‐RIPK3, t‐RIPK3, p‐MLKL and t‐MLKL. b) The protein expression of NLRP3 in GMEC was detected by IF. Scale bar: 50 µm. c) The protein expression of NLRP3 in GMEC organoids was detected by IF. Scale bar: 100 µm. d) Primary GMEC and gene‐edited GMEC were treated with 5 µg mL⁻¹ LPS and 10 µg mL⁻¹ LPS for 12 h, and then the culture medium was replaced for 12 h to detect the proportion of apoptosis and necrosis (n = 3 per group). Scale bar: 100 µm. e) Primary GMEC and gene‐edited GMEC were treated with 5 µg mL⁻¹ LPS and 10 µg mL⁻¹ LPS for 12 h, and then the culture medium was replaced for 12 h to detect apoptosis ratio (n = 3 per group). f) Under inflammatory conditions, primary GMEC were treated with Z‐VED, MCC950 and Nec‐1 inhibitors to inhibit the activation of PANoptosis and to detect the protein expression of ZO‐1 and Occludin.

Figure 9

A overview chart for mechanism of alleviating mastitis and BMB damage by GED goats with high expression of LYZ. a) According to the regulatory sequence gene editing breeding strategy proposed in this study, GED goats were created by targeted integration of IRS into the LYZ promoter region using the ISDar2‐TnpB system. b) Isolation of GMEC from GED goats for culture and establishment of GMEC organoids. c) E.coli infection of the mammary gland of GED goats increases LYZ expression, which inhibits the activation of PANoptosis, decreases SCC and proinflammatory factor expression in milk, and increases ZO‐1 and occludin expression, thereby alleviating mastitis severity and BMB damage. d) In vitro, a mastitis model was established by treating GMEC with LPS, and the TLR4/MyD88/NF‐κB signaling pathway was activated. e) When the TLR4/MyD88/NF‐κB signaling pathway is activated, a large amount of pro‐inflammatory factors are produced and secreted extracellularly. f) In an in vitro GMEC mastitis model, the PANoptosome can be activated. g) Under inflammatory conditions, IRS upregulates LYZ expression in gene‐edited GMEC. h) In an in vitro GMEC mastitis model, the PANoptosis can be activated. i) Activation of PANoptosis disrupts membrane integrity and permeability. j) Activation of PANoptosis results in the release of a large number of pro‐inflammatory factors. k) In an in vitro GMEC mastitis model, LYZ can exert an anti‐inflammatory role by regulating the expression of HMGB1.