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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Genomics and Improvement Laboratory » Research » Publications at this Location » Publication #372804

Research Project: Improving Dairy Animals by Increasing Accuracy of Genomic Prediction, Evaluating New Traits, and Redefining Selection Goals

Location: Animal Genomics and Improvement Laboratory

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

item Cole, John
item Bickhart, Derek
item Hutchison, Jana
item Null, Daniel
item ORTEGA, M - University Of Missouri

Submitted to: European Association of Animal Production Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 3/16/2020
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: 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, is described. Holstein Haplotype 2 (HH2), which causes early embryonic death, was used to demonstrate the approach. The HH2 haplotype 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. The frameshift was confirmed in a separate group of Holsteins from Run7 of 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 proven high-fertility semen. 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. Editing efficiency among three replicates was 26% for biallelic edits and 32% for mono-allelic edits based on visualization of PCR fragments on an agarose gel. The percentage of putative zygotes that cleaved was determined at 3 d of development (d 0 = day of insemination) and blastocyst rate was estimated at 8 d. 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. The system described can be used to validate causal variants for deleterious mutations that act early in development.