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

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 P_C Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (P_Cp) 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 P_C Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the P_C 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.

Fig. 1. Dendrogram of the phylogenetic relationship (IBS distance) among the sequences analyzed in this study.

Less similar sequences have clade branch points closer to the center of the circle. Genome-edited polled bull (RCI002, black) and progenitor cell line (CL2122.org, black); Horned Hereford bulls (purple); Holstein bull (pink); Horned Hereford cows (brown); calves (blue); unrelated genome-edited bull (RCI001.org, red) and progenitor cell line (CL2120.org, red). The genome-edited polled bull sequence is represented twice; once (RCI002) from sequencing performed as part of this study and once (RCI002.org) as the original sequence reported by Carlson et al..

Fig. 2. Offspring of the genome-edited polled bull and controls.

Shown are the six offspring and six contemporary controls at <3 months of age (before any horn development) and their parents. a, Study group GH.H.: the genome-edited polled bull (RCI002) was bred to Horned Hereford cows (RC.dams1–6) and produced six polled offspring (RC.calves1–6). b, Study group H.H.: Horned Hereford bulls (HH.sire1 and HH.sire23) bred to Horned Hereford cows (HH.dams1–3) by artificial insemination or natural service produced three horned offspring (HH.calves1–3). c, Study group Ho.H.: the Horned Holstein sire (HO1) of the genome-edited polled bull in a was bred to Horned Hereford cows (HO1.dams1–3) by artificial insemination and produced three horned offspring (HO1.calves1–3). P_C designates the Celtic POLLED allele (dominant), P_C* designates the additional introgression of the HDR donor plasmid along with the P_C Celtic allele and p designates the wild type HORNED allele (recessive). Offspring are labeled as male or female by blue and pink symbols, respectively. All pictures are of the actual animals, with the exception of the two Horned Hereford bulls, for which the images are representative.

Fig. 3. PCR results for the genotypes of the offspring of the genome-edited bull (RC.calves1–6) and three of the contemporary Horned Hereford controls (HH.calves1–3).

Homozygous polled (Polled, 591 bp), homozygous horned (Horned, 389 bp) and negative PCR controls are shown at the right. The offspring of the genome-edited polled bull are heterozygous POLLED and the Horned Hereford controls are homozygous HORNED. This PCR was carried out for the 28 animals sequenced in this study.

Fig. 4. The number of SNP variants relative to the ARS-UCD1.2 bovine reference genome derived from a Hereford cow.

The Hereford cow was L1 Dominette 01449 (ref. ). Males (squares) and females (circles) are shown in four study groups. a, GH.H.: genome-edited polled bull (RCI002, black) was bred to Horned Hereford cows (RC.dams1–6, brown) and produced six polled offspring (RC.calves1–6, blue). b, H.H.: Horned Hereford bulls (HH.sire1 and HH sire23, purple) were bred to Horned Hereford cows (HH.dams1–3, brown) and produced three horned offspring (HH.calves1–3, blue). c, Ho.H.: Horned Holstein sire (HO1, pink) was bred to Horned Hereford cows (HO1.dams1–3, brown) by artificial insemination and produced three horned offspring (HO1.calves1–3, blue). d, Carlson: original sequences of cell lines (CL2122.org (black) and CL2120.org (red)) that were edited to produce bulls, RCI002.org (black) and RCI001.org (red), respectively, as reported by Carlson et al.. RCI002 and RCI002.org are data from the same genome-edited bull sequenced by two different sequencing laboratories in different years.

Fig. 5. The alleles of the bovine POLLED locus.

a,b, Difference between the wild type HORNED allele (a) and naturally occurring P_C POLLED allele (b) within the 1.6 kb HDR template sequence (Carlson et al.) at the POLLED locus. The 212-bp repeat sequence (purple) is duplicated in the naturally occurring P_C POLLED allele and replaces the 10-bp (CTGGTATTCT) orange sequence (*) in the wild type HORNED allele. btHP-F1/btHP-R2 are PCR primers used by Carlson et al. and for our screening PCR in Fig. 3. c,d, The genome-edited bull RCI002 was a compound heterozygote carrying allele (c) the exact same sequence as the naturally occurring P_C POLLED allele and allele (d) that included both the pCR2.1 plasmid sequence (yellow) and a duplication of the Pc HDR template (red). topoIF/M13R and M13F/topoIR are PCR primer pairs.