Haplotype- and sequence-based identification of a deletion associated with early embryonic loss in Holstein cattle and functional validation using CRISPR-Cas9 knockouts

D.M. Bickhart1, J.B. Cole*,2, J. L. Hutchison2, D.J. Null2, and M.S. Ortega3

1Cell Wall Biology and Utilization Research Laboratory, U.S. Dairy Forage Research Center, Agricultural Research Service, United States Department of Agriculture, Madison, WI, 53706
2Animal Genomics and Improvement Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705
3Division of Animal Sciences, College of Agriculture, Food, and Natural Resources, University of Missouri, Columbia, 65211

2020 J. Dairy Sci. (?)
© American Dairy Science Association, 2020. All rights reserved.
Individuals may download, store, or print single copies solely for personal use.
Do not share personal accounts or passwords for the purposes of disseminating this article.


We present a comprehensive framework for identification and validation of genetic defects, including haplotype-based detection of defects, selection of variants from sequence data, and in vitro validation using CRISPR-Cas9 knockout embryos. Holstein Haplotype 2 (HH2), which causes early embryonic death, was used to demonstrate the approach. HH2 was identified using a deficiency-of-homozygotes approach and confirmed to have undesirable effects on conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the coverage of unique haplotypes. Three variants concordant with the haplotype calls were found in HH2: a high-priority frameshift mutation resulting from a deletion, and two low-priority variants (1 synonymous variant, 1 premature stop codon). The frameshift was confirmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. Intraflagellar protein 80 (IFT80)-null embryos were generated by truncating the IFT80 transcript at exon 2, using two guide-RNAs annealed to Cas9 mRNA. Abattoir-derived oocytes were fertilized in vitro with a proven high-fertility sire. Embryos were injected at the one-cell stage either with CRISPR-Cas9 complex (n=100) or Cas9 mRNA (control, n=100) before return to culture, and replicated 3 times. IFT80 is activated at the 8-cell stage, and IFT80-null embryos arrested at the 8-cell stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. A frameshift in IFT80 on chromosome 1 at 107,172,615 bp (p.Leu381fs) disrupts wnt and hedgehog signaling, and is responsible for the death of homozygous embryos.