Precise gene editing paves the way for derivation of Mannheimia haemolytica leukotoxin-resistant cattle

Abstract

Signal peptides of membrane proteins are cleaved by signal peptidase once the nascent proteins reach the endoplasmic reticulum. Previously, we reported that, contrary to the paradigm, the signal peptide of ruminant CD18, the β subunit of β₂ integrins, is not cleaved and hence remains intact on mature CD18 molecules expressed on the surface of ruminant leukocytes. Leukotoxin secreted by Mannheimia (Pasteurella) haemolytica binds to the intact signal peptide and causes cytolysis of ruminant leukocytes, resulting in acute inflammation and lung tissue damage. We also demonstrated that site-directed mutagenesis leading to substitution of cleavage-inhibiting glutamine (Q), at amino acid position 5 upstream of the signal peptide cleavage site, with cleavage-inducing glycine (G) results in the cleavage of the signal peptide and abrogation of leukotoxin-induced cytolysis of target cells. In this proof-of-principle study, we used precise gene editing to induce Q(‒5)G substitution in both alleles of CD18 in bovine fetal fibroblast cells. The gene-edited fibroblasts were used for somatic nuclear transfer and cloning to produce a bovine fetus homozygous for the Q(‒5)G substitution. The leukocyte population of this engineered ruminant expressed CD18 without the signal peptide. More importantly, these leukocytes were absolutely resistant to leukotoxin-induced cytolysis. This report demonstrates the feasibility of developing lines of cattle genetically resistant to M. haemolytica-caused pneumonia, which inflicts an economic loss of over $1 billion to the US cattle industry alone.

Keywords: Mannheimia haemolytica; cattle; cloning; leukotoxin-resistant.

Fig. 1.

Development of bovine fetal fibroblasts containing the Q(‒5)G substitution in both alleles of CD18. (A) Schematic representation of ZFN-mediated gene editing of bovine ITGB2. (B) ZFN-mediated induction of the Q(‒5)G substitution. The pair of ZFNs were designed to cleave bovine ITGB2 in the vicinity of the codon targeted for editing (red fonts). Sequences bound by each ZFN are underlined in red. ITGB2 domain targeted for FokI-cleavage is in blue fonts. (C) Cel-1 assay. Lanes T and U show the digestion products from the ZFN-transfected and ZFN-untransfected cells, respectively. Lane MM represents molecular weight markers. (D) PCR-RFLP assay. The ∼1.8-kb and ∼0.9-kb bands represent the undigested and digested products, respectively. In this figure, lanes 1, 2, 4, and 8 show clones that are homozygous, whereas lane 5 shows a clone that is heterozygous for the mutation. Lanes 3, 6, and 7 show clones without any modification. (E) Sequencing analysis. Sequencing data shows the presence of glycine-encoding GGA codon instead of the glutamine-encoding CAG codon.

Fig. 2.

Leukocytes from the gene-edited fetus express CD18 without the signal peptide. (A) Leukocytes from the gene-edited fetus contain the Q(‒5)G substitution in both alleles of CD18. Sequencing data shows the presence of glycine-encoding GGA codon in the CD18 DNA from the gene-edited fetus (a1) and glutamine-encoding CAG codon in the CD18 DNA from the wild-type CD18 DNA (a2). (B) CD18 expression on leukocytes from the gene-edited fetus is comparable to that on leukocytes from a wild-type calf. CD18 expression on the leukocytes from the gene-edited fetus (b1) or a wild-type calf (b2) was determined by flow cytometry. The blue and red histograms represent binding of anti-CD18 antibodies and isotype-matched control antibodies, respectively. The black histogram represents unstained cells. (C) CD18 expressed on leukocytes from the gene-edited fetus lacks the signal peptide. The presence of signal peptide on CD18 expressed on the leukocytes from the gene-edited fetus (c1) or a wild-type calf (c2) was determined by flow cytometry. The magenta and blue histograms represent binding of chicken anti-signal peptide serum and unimmunized chicken serum, respectively. The red histogram represents unstained cells. Leukocytes from the wild-type calf expressed CD18 containing the signal peptide, whereas the leukocytes from the gene-edited fetus expressed CD18 without the signal peptide. Results of one representative experiment of two are shown.

Fig. 3.

Leukocytes from the gene-edited fetus are resistant to M. haemolytica leukotoxin-induced cytolysis. Leukocytes from the gene-edited fetus and a wild-type calf were subjected to the MTT dye-reduction cytotoxicity assay with serial dilutions of M. haemolytica leukotoxin. Leukocytes from the wild-type calf (blue line) were lysed in a concentration-dependent manner, whereas the leukocytes from the gene-edited fetus (red line) were not lysed even by the undiluted leukotoxin. All data are expressed as mean ± SD (n = 3).