Recessive alleles represent genetic risk in populations that have undergone bottleneck events. We present a comprehensive framework for identification and valida-tion of these genetic defects, including haplotype-based detection, variant selection from sequence data, and validation using knockout embryos. Holstein haplotype 2 (HH2), which causes embryonic death, was used to demonstrate the approach. Holstein haplotype 2 was identified using a deficiency-of-homozygotes approach and confirmed to negatively affect conception rate and stillbirths. Five carriers were present in a group of 183 sequenced Holstein bulls selected to maximize the cov-erage of unique haplotypes. Three variants concordant with haplotype calls were found in HH2: a high-priority frameshift mutation resulting, and 2 low-priority vari-ants (1 synonymous variant, 1 premature stop codon). The frameshift in intraflagellar 80 (IFT80) was con-firmed in a separate group of Holsteins from the 1000 Bull Genomes Project that shared no animals with the discovery set. IFT80-null embryos were generated by truncating the IFT80 transcript at exon 2 or 11 us-ing a CRISPR-Cas9 system. Abattoir-derived oocytes were fertilized in vitro, and zygotes were injected at the one-cell stage either with a guide RNA and CAS9 mRNA 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 this stage of development, which is consistent with data from mouse hypomorphs and HH2 carrier-to-carrier matings. This 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.
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