Breeding of polled calves

Semen from a genome-edited polled bull (RCI002)2 was collected, cryopreserved and used to artificially inseminate ten estrus-synchronized Horned Hereford cows. This bull originated from the University of Minnesota dairy crossbreeding program and is known to be 62.5% Holstein, 25% Montbelliarde and 12.5% Jersey. Six pregnancies resulted, with one female and five male calves born in September 2017. This pregnancy rate of 60% is comparable to those reported under similar estrus-synchronization and artificial insemination protocols9. Contemporary controls consisted of purebred Horned Hereford calves (two females and one male born in September 2017). Horned Hereford cows were also bred to the Holstein sire (HO1) of RCI002 by artificial insemination and three calves (one female, two males) were born in December 2017. Figure 1 shows a dendrogram of the identity by state (IBS) distance among the DNA sequences from the 28 cattle (pictured in Fig. 2) involved in this study along with the original sequences from Carlson et al.2. Genetic testing verified the parentage of each calf (Methods).

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 bull2 (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.2. Full size image

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. Full size image

Sequencing data from the same individual performed at different sequencing laboratories (that is, RCI002 and RCI002.org) differed more than the sequences of an edited animal and its unedited progenitor cell line sequenced at the same time and location (for example, CL2122.org and RCI002.org) (Fig. 1). In some cases, the Horned Hereford dams were closely related and cluster together. For example, HO1.dam1 and HO1.dam3 (upper left) are full siblings, and RC.dam2, who groups closely with them, is their half-sibling based on pedigree records.

Assessment of calf health

The calves were born without incident, with the exception of one Holstein (HO1) × Hereford control calf that was breech and required veterinary intervention at birth. A comprehensive veterinary physical examination was performed on all of the calves at approximately one week of age, including palpation for the presence of horn buds. Horn buds were not present in calves from the genome-edited sire, but were present in Hereford control calves and Holstein × Hereford calves (Fig. 2). All routine physical parameters were within normal limits and comparable between the offspring of the genome-edited polled bulls and control calves. All bull calves had two descended testicles, with the exception of one of the offspring from the genome-edited polled bull (RC.calf6) that had one descended testicle and one cryptorchid testicle external to the inguinal ring, above the neck of the scrotum. Complete blood counts and blood chemistry analyses were performed, with results comparable across all groups of calves.

Additional veterinary physical exams, evaluating the same metrics, were performed at approximately 8 and 12 months of age. All calves were healthy and all parameters were within normal limits. In addition, bull calves in the genome-edited offspring and control offspring groups underwent breeding soundness examinations at 15 months of age, following the standards set out by the Society of Theriogenology10. Four bulls from the genome-edited offspring group passed and were classified as satisfactory potential breeders, while one bull (RC.calf6) was unsatisfactory due to an undescended (cryptorchid) testicle. All control bulls were deemed satisfactory potential breeders. No calves in any group had any significant health events during the study timeframe. At the completion of this study, the bull RCI002 and his five male offspring were euthanized and incinerated as their intentional genome edits were unapproved animal drugs8, and therefore could not be marketed to enter the food supply.

Assessment of POLLED genotype

Blood samples were collected, DNA extracted and PCR performed to test for POLLED and HORNED alleles as described2. The six offspring of the genome-edited polled bull (RC.calves1–6) were heterozygous for POLLED (P c p). The Horned Hereford control calves (HH.calves1–3) were homozygous horned (pp, Fig. 3 and Supplementary Fig. 1) as were the offspring of the Holstein sire (data not shown). The Horned Hereford cows had their horns removed physically, which is why no horns are visible in Fig. 2. Records for RC.dam1 indicate that she was disbudded along with the rest of her herdmates, but she is heterozygous P C p by PCR and therefore was naturally polled.

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. Full size image

Assessment of horned phenotype

By the 8-month exam, the purebred control Horned Hereford calves (HH.calves1–3) and the Holstein × Hereford calves (HO1.calves1–3) had developed horns, as expected. The calves sired by the genome-edited polled bull had not developed horns (Supplementary Fig. 2); however, the bull calves did develop small scurs (Supplementary Fig. 3). Scurs, corneous growths that can be of varying sizes and develop in the same area as horns but are not firmly attached to the skull, are a common occurrence in males heterozygous for POLLED11, so this result is not surprising or outside of normal parameters. The heifer calf did not develop scurs. Scurs map to a separate genetic locus from the POLLED locus, but the exact causal mutation remains unknown12. At the time of writing, the one remaining female calf is 23 months old and still has not developed horns.

Assessment of fetal microchimerism

To evaluate whether fetal cells potentially crossed the placental barrier to the surrogate dams (fetal microchimerism), blood samples were taken from the dams 1 month before birth and at weeks 1, 2, 3, 4 and 5. DNA was extracted and assayed by quantitative PCR (qPCR) for HORNED, POLLED, a Y chromosome marker and a housekeeping gene (data not shown). All dams showed the presence of the HORNED allele, as expected. RC.dam1 showed the presence of the HORNED allele and the POLLED allele consistent with PCR results for this dam that indicate heterozygosity for the POLLED allele. None of the dams that carried male offspring showed the presence of the Y chromosome marker. The results did not show any transfer of the POLLED allele from the genome-edited polled sire offspring to the blood of the dams.

Assessment of genomic variation

The genome-edited bull’s (RCI002) offspring were compared to matching controls with reference to the ARS-UCD1.2 bovine genome sequence (https://www.ncbi.nlm.nih.gov/assembly/GCF_002263795.1/), derived from a Hereford cow13, to determine whether the number of single nucleotide polymorphisms (SNPs), indels and Mendelian transmission rates were skewed in any of the study groups (GH.H versus H.H versus Ho.H).

Variant calling and variant statistics

GATK variant calling initially identified 17,758,947 variants. A subsequent quality filtration identified 14,155,980 variants as trusted. The numbers of variants (in the range of 4–7 million SNPs (Fig. 4) and 80,000–100,000 indels per individual) were comparable in all animals. There was an obvious result of fewer variants found when comparing the sequence of purebred Horned Herefords (H.H family) to the reference Hereford genome, as compared to sequences from purebred Holstein (HO1) or the Holstein cross (RCI002) bull, and offspring sired by these bulls (Fig. 4).

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. 13). 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.2. RCI002 and RCI002.org are data from the same genome-edited bull sequenced by two different sequencing laboratories in different years. Full size image

Assessment of Mendelian errors

Biallelic variants (14,084,653) achieved a 99.8% genotyping rate and were included in further analyses. Another subset of variants was also selected by exclusion of 218,070 variants with genotype rate <95% and 2,537,388 variants with minor allele frequency <5%. The breakdown of heterozygous, compound heterozygous and homozygous mutants for each animal as compared to the reference genome is detailed in Supplementary Table 1. Four families with 12 meiotic divisions were tested for the number of errors according to the expected rate of Mendelian transmission (Table 1). With both datasets, the average rate of the errors in each meiotic division was 1.0% per variant (±0.2) with insignificant differences between the three studied groups (two one-way analysis of variance (ANOVA) d.f. = 2; P = 0.078, F = 3.43; P = 0.149, F = 2.369). Mendelian error rates in 10 kilobase regions accounting for a high proportion of inherited errors did not differ in range among the study families (Supplementary Fig. 4). ANOVA for the average error rates per study group (d.f. = 2, F = 61.101) showed no difference between GH.H. and Ho.H. groups (P = 0.897); however, both groups were significantly different from the H.H. group (P < 0.001; Supplementary Fig. 4). The 171 regions with consistently high error rates (>1 error per kb) in all three study groups were most prevalent on Chromosomes 12 and 23, and are listed in Supplementary Table 2.

Table 1 Mendelian error rates of n = 12 biologically independent sire/dam/offspring trios in four families Full size table

Assessment of insertion stability

A sequence baiting approach was used to investigate whether the 212 base pair repeat of the P C POLLED allele was inserted anywhere in the genome other than the expected position. The sequence inserted in the correct location is expected to cause a duplication of an internal 5′ 212 bp in the cattle reference genome (Fig. 5a,b). If the sequence is appropriately inserted in, and only in, the expected position, all reads generated from the sequence of this insertion locus should be categorized into one of three classes when mapped back to the ARS-UCD1.2 bovine reference genome sequence: (1) reads mapping perfectly to the internal repeat or its 5′ junction with the reference genome, (2) reads mapping to the 3’ end of the internal repeat with a 16-bp deletion and (3) reads mapping with supplementary alignment to this locus but align perfectly over the junction between the two repeats in the reference genome sequence amended to have the insertion sequence (Fig. 5c). In this approach, we selected any sequence that shared at least 25 bp of the 212 bp of the P C polled allele to find any possible degenerate or chimeric versions of the insertion sequence.

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.2) 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.2 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. Full size image

The sequence baiting approach found that all reads generated from the insertion sequence and the surrounding edges matched one of the three expected classes, with the exception of a single read. That read only mapped to the original and expected loci with supplementary alignments. Revised exact alignment of the read showed that it belonged to the third category above, but had many sequencing errors that prevented the direct alignment to the expected locus (Supplementary Fig. 5). Only those animals carrying one or two copies of the P C POLLED allele had reads that aligned perfectly to class c, meaning they aligned around the insertion position in the ARS-UCD1.2 bovine reference genome sequence at the predicted insertion sequence. The P C POLLED allele did not insert anywhere in the genome other than the expected position.

Assessment for the presence of plasmid sequence