Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating

Abstract

Poultry is the most abundant livestock species with over 60 billion chickens raised globally per year. The majority of chicken are produced from commercial flocks, however many indigenous chicken breeds play an important role in rural economies as they are well adapted to local environmental and scavenging conditions. The ability to make precise genetic changes in chicken will permit the validation of genetic variants responsible for climate adaptation and disease resilience, and the transfer of beneficial alleles between breeds. Here, we generate a novel inducibly sterile surrogate host chicken. Introducing donor genome edited primordial germ cells into the sterile male and female host embryos produces adult chicken carrying only exogenous germ cells. Subsequent direct mating of the surrogate hosts, Sire Dam Surrogate (SDS) mating, recreates the donor chicken breed carrying the edited allele in a single generation. We demonstrate the introgression and validation of two feather trait alleles, Dominant white and Frizzle into two pure chicken breeds using the SDS surrogate hosts.

Fig. 1. iCaspase9-mediated ablation of the germ cell lineage.

a CRISPR/Cas9-mediated recombination of an iCaspase9 and GFP reporter gene into the final coding exon of the DAZL locus. Red arrow indicates the guide target. b 500 PGCs targeted with a human iCaspase9 or a chicken iCaspase9 (aviCaspase9) transgene were cultured in the presence of different concentrations of the B/B dimerisation compound for 10 days. Data are presented as the mean ± SD, n = 3 (n = 2 for 100 nM). One-way ANOVA, *P < 0.05, ***P < 0.001 with respect to Dazl-GFP cells. c Day-10 embryonic gonads were examined for expression of GFP and SSEA1 immunofluorescence, n = 4. d Control and (e) B/B-treated iCaspase9 G₂ embryos imaged at day 10 of incubation. Arrows indicate the embryonic gonads. *, autofluorescence in the underlying mesonephros. n = 5 for each genotype. Scale bar, 100 μm.

Fig. 2. Genome edit to remove the DOW allele from a White leghorn chicken breed.

a PMEL17 locus containing the DOW allele (WAP insertion) within the 10th exon which encodes for the PMEL17 transmembrane domain (amino acids in black). Genome-edited PMEL17 locus removing the WAP (highlighted green). Genome-edited second allele creating a five amino acid Dun-like deletion (deleted amino acids highlighted yellow). The five amino acids deleted in the Dun allele are highlighted in blue. b, d Wild-type WL Line 6 chicks and adults. c, e PMEL17 edited chicks and adults; females (standing) were barred and speckled feathers, males (sitting) were white with black dots. All photographs of live birds at The Roslin Institute.

Fig. 3. Schematic of SDS mating.

PGCs are isolated and propagated in vitro from a pure chicken breed. After genome editing and clonal isolation, the male and female PGCs are mixed with the B/B compound and injected into iCaspase9 surrogate host embryos. The embryos are hatched, raised to sexual maturity then mated. Laid eggs and hatched offspring are from the donor breed of interest and contain the desired genome edit. Drawing by MJM.

Fig. 4. PC analysis of G1 offspring from iCaspase9 surrogate host birds.

DNA samples were genotyped on a 66 K SNP chip and analysed for PCs. Three chicken breeds were analysed: the brown layer breed which includes the iCaspase9 hosts, the LSX breed, and the WL Line 6 breed. Offspring from iCaspase9 hosts injected with LSX PGCs (grey) clustered with LSX control birds. Offspring from iCaspase9 surrogate hosts injected with WL Line 6 PGCs (green) clustered with WL Line 6 control birds.

Fig. 5. Genome edit to introduce the FRZ allele into the LSX chicken breed.

a KRT75 locus surrounding the FRZ mutation. A single guide (blue) that targets cleavage 12 bp from the intended deletion was transfected along with a ssODN containing 50 bp homology on each side of the deletion. A sequencing chromatogram shows the bi-allelic deletion in female LSX PGCs. b PCR analysis of KRT75-edited LSX PGCs and G₁ offspring from iCaspase9 hosts. The wild-type locus produces a PCR product of 657 bp. The edited locus produced a PCR product of 573 bp, n = 11. c, e Control 6-week-old offspring and wing (18 weeks) from LSX birds. n = 10/10 female birds. d, f FRZ heterozygote LSX G1 offspring (6 weeks) and wing (18 weeks) displaying crumpled flight feathers, n = 5 of 5 female birds. All photographs of live birds at The Roslin Institute.