‘Natural AI’: towards generating absolute transmitter rams by genome editing and embryo complementation
Zachariah L. McLean, Russell Grant Snell, Björn Oback · Figshare · 2018
Mammalian male germline transmission depends on the gene DAZL, which encodes an RNA-binding protein, and DAZL homozygous null males (-/-) are sterile. We aim to produce chimeric ‘absolute transmitter’ rams whose own germline has been replaced with that of a genomcially selected elite donor embryo. This will be achieved by genetically disabling spermatogenesis and complementing the vacant sperm niche with germline-competent embryonic donors. For proof-of-concept, DAZL-/- somatic cell nuclear transfer (SCNT) cloned male host embryos and animals were produced by genome editing. We employed CRISPR/Cas9 genome editors to disrupt DAZL in male ovine fetal fibroblasts (OFFs). A single-stranded homology-directed repair (HDR) template was designed to mediate a small insertion, introducing a stop codon and Taq1 restriction site. Allelic discrimination assays on 48 genome edited single cell clones showed three distinct populations, namely 67% wildtype, 10% homozygous and 23% heterozygous DAZL mutants. DNA sequencing and Taq1 digest confirmed biallelic editing for all DAZL-/- clones. Proven cell clones were used as donor cells for SCNT and used to generate embryos for transfer into surrogate recipients. Following lambing, newborn edited animals were analysed for their testis phenotype. Testis cords contained histologically normal somatic support cells but lacked spermatogonia. This demonstrates that genetically sterilised male embryos could serve as suitable hosts for subsequent aggregation with wildtype NT donor embryos to colonise the vacant niche. Successful developmental compensation would provide an alternative to artificial insemination (AI) in extensive farming systems, accelerating genetic gain through teams of absolute transmitter rams that rapidly disseminate their high-value donor haplotype by ‘natural AI’.