Department of Animal Science, University of CA - Davis, Davis, CA, USA.
Department of Population Health and Reproduction, School of Veterinary Medicine, University of CA - Davis, Davis, CA, USA.
Sci Rep. 2022 Feb 8;12(1):2067. doi: 10.1038/s41598-022-06118-6.
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (P allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.
去角是奶牛养殖业中的常见做法，但引起了动物福利方面的关注。一个自然发生的遗传突变（P 等位基因）由一个包含 212bp 重复 DNA 序列的组成，取代了多乳头基因座的 10bp 序列，与牛的无角表型（无角）有关。为了验证仅 10bp 缺失就足以导致无角的假设，优化了 CRISPR-Cas9 双引导 RNA 方法，以删除包括 10bp 序列在内的 133bp 区域。比较了在不同受精后时间（hpi）（6、8 和 18 hpi）以及体外转录（IVT）与合成 gRNA 注射核糖核蛋白复合物的时间。与 8 hpi（12%）和 18 hpi（7%）相比，6 hpi 注射的胚胎的缺失率（53%）明显更高，与 IVT gRNA（53%）相比，合成 gRNA 的缺失率（84%）明显更高。进行了胚胎移植，并在妊娠 3 至 5 个月时收获了牛胎儿。所有胎儿在靶位点都发生了突变，其中 7 个中有 2 个发生了双等位基因缺失，但它们显示出角芽发育，表明仅 10bp 缺失不足以导致无角表型。
