Table1_Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos.XLSX

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

基因组编辑（Genome editing）为改良当前肉牛育种策略提供了全新机遇，可通过靶向引入天然序列变异加速遗传增益。该技术可借助编辑工具诱导基因组切割后，在修复模板存在的条件下利用同源定向修复（homology-directed repair）机制实现。将基因组编辑工具导入受精卵并在胚胎中开展编辑，其优势在于可正常发育为活畜，且与当前基于胚胎的育种改良流程兼容。
本研究旨在探索引入已知来自前黑素小体蛋白17（pre-melanosomal protein 17, PMEL）与催乳素受体（prolactin receptor, PRLR）基因的序列变异的潜力，以培育非嵌合型编辑胚胎，并将其完全精准转化为目标基因型。向牛受精卵注射向导RNA（guide RNA, gRNA）/Cas9编辑工具以在PMEL基因中引入3 bp缺失变异，最高可获得11%的完全转化胚胎。使用转录激活因子样效应物核酸酶（transcription activator-like effector nucleases, TALEN）并通过质粒递送时，转化效率可提升至最高48%。针对多种已知PRLR序列变异、不同修复模板设计，以及以DNA、RNA或核糖核蛋白（ribonucleoprotein, RNP）形式递送的三种gRNA/Cas9编辑工具进行测试，最高可实现8%的完全转化效率。此外，本研究开发了一种基于活检的非嵌合胚胎筛选策略，有望仅培育携带精准编辑位点的非嵌合型活畜。