200 words) 13

Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knock-ins into livestock genomes, followed by cloning. Editing embryos directly to achieve targeted gene knock-ins is inefficient, especially for introducing large DNA sequences. Here we report using a one-step method to produce a gene knock-in bull calf by cytoplasmic microinjection of CRISPR/Cas9 reagents into a bovine embryo. In vitro fertilized one-cell bovine zygotes were injected with a gRNA/Cas9 ribonucleoprotein complex and homology mediated end joining donor template containing the sex determining region Y (SRY) gene, the green fluorescent protein (gfp) reporter gene driven by the SV40 promoter, and one kilobase homology arms targeting the H11 safe harbor locus on bovine chromosome 17. Seven-day blastocysts were evaluated using fluorescent microscopy, and nine green fluorescent embryos were transferred to synchronized recipients. Ultrasound evaluation at 35 days revealed one pregnancy. In April 2020, a healthy 50 kg male calf was born. DNA was extracted from placenta, blood and a fibroblast line derived from the calf and analyzed for SRY-GFP knock-in, as well as genotypic sex. PCR and Sanger sequencing revealed the biallelic edit of the target location on chromosome 17, with the insertion of three or seven copies of the SRY-GFP construct in addition to donor plasmid backbone, or a 26 base pair insertion, and an XY genotype. Future analysis of the XX offspring inheriting the SRY gene on chromosome 17 from this knock-in bull will reveal whether inheritance of the bovine SRY gene is sufficient to trigger the male developmental pathway in cattle.