Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

Amy E Young,Joseph R Owen,Bret R Mcnabb,Josephine F Trott,C Titus Brown,Alison L Van Eenennaam,Tamer A Mansour

doi:10.1038/s41587-019-0266-0

Amy E Young, Joseph R Owen + Show 5 more

Open Access

https://doi.org/10.1038/s41587-019-0266-0

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Abstract

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around ‘intentional genomic alterations’ and future regulatory actions regarding genome-edited animals.

Highlights

Edits will likely need to be introduced into multiple elite founder animals to prevent genetic bottlenecks[3]
Others have reported on Whole-genome sequencing (WGS) of trios of genomeedited (CRISPR/Cas9) knockout livestock produced through cytoplasmic injection (CPI) of guide RNA and Cas[9] into one-cell-stage zygotes
A third study used an unbiased WGS on two genome-edited calves produced by a targeted gene knockout of beta-lactoglobulin using CPI of a homology-directed repair (HDR) donor plasmid and transcription activator-like effector nucleases (TALENs) into early zygotes[7]

Summary

Introduction

Edits will likely need to be introduced into multiple elite founder animals to prevent genetic bottlenecks[3]. A third study used an unbiased WGS on two genome-edited calves produced by a targeted gene knockout of beta-lactoglobulin using CPI of a homology-directed repair (HDR) donor plasmid and TALENs into early zygotes[7]. These calves were free of any TALENmediated off-target mutations or donor plasmid integration events. The calves produced as part of the current study are, to our knowledge, the first reported offspring of a genome-edited bull These data will help inform regulatory agencies as they formulate processes to regulate genome-edited livestock. Appropriate regulation is of pivotal importance if this technology is to have a role in commercial livestock production, especially in light of the 2017 United States Food and Drug Administration’s Draft Guidance for Industry no. 187, entitled ‘Regulation of Intentionally Altered Genomic DNA in Animals’[8], which judges intentional DNA alterations as new animal drugs

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