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

Sudarvili Shanthalingam, Ahmed Tibary, Jonathan E. Beever, Poothapillai Kasinathan, Wendy C. Brown, Subramaniam Srikumaran

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)13186-13190
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number46
DOIs
StatePublished - Nov 15 2016

Keywords

  • Cattle
  • Cloning
  • Leukotoxin-resistant
  • Mannheimia haemolytica

ASJC Scopus subject areas

  • General

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