Edits of AR in porcine blastocysts after CRISPR Cas9 editing of zygotes

The CRISPR Cas9 technology offers an opportunity to evaluate physiological responses to deletion of individual genes in livestock species, expanding our understanding of animal biology. Testing efficacy of individual guides used for gene deletion is desirable before the time-consuming embryo transfers planned to generate the desired model animals. These experiments were designed to test efficacy of guides to delete expression of the androgen receptor (AR) in pig zygotes and to characterize the resulting deletions. Two guides targeting exon 2 (Ensembl Sus Scrofa 11.1) were designed. Following electroporation of these guides into in vitro generated porcine zygotes, embryos were cultured to the blastocyst stage and the DNA sequenced. Over 80% of blastocysts produced following electroporation of the two guides targeting exon 2 had deletions. Deletions were biallelic. Although blastocysts might contain edits from only one of the guides, the majority of blastocysts had large deletions resulting from the combined effect of both guides.

Methods
Cumulus oocyte complexes were aspirated from 3-5 mm follicles on pig ovaries obtained from a commercial abbatoir, rinsed and matured in vitro for 40-44 hours at 38.5 o C. The 100 μL drop containing oocytes and approximately 1000 washed, partially capacitated sperm/oocyte was incubated for 5-6 hours in 5% CO2 in air. Zygotes were electroporated in Opti-MEM™ (Gibco) with the CRISPR guide and Cas9 protein 9 hours after insemination. Following culture of electroporated zygotes to the blastocyst stage, individual blastocysts were lysed in 10 µL of QE09050 buffer (Biosearch Technologies, UK), vortexed, centrifuged, and incubated at 65°C (6 minutes) and 98°C (2 minutes) followed by storage at -17o C. DNA was amplified during nested rounds (2) of PCR, the PCR products were separated on a 0.8% agarose gel, bands excised, DNA was extracted (QIAquick Gel Extraction Kit, Qiagen #28704) and submitted for sequencing. Sequences were aligned with SnapGeneâ using the Sus Scrofa 11.1 genome. The sequences around the target sites of the two CRISPR guides from 9 representative blastocysts are presented in Figure 1. With electroporation with the combination of the two CRISPR guides targeting exon 2 of the porcine AR (targets outlined in red in the reference sequence), the vast majority of blastocysts have large deletions and a blastocyst has a 6 bp deletion. The last edit might only eliminate two amino acids in the transcript or potentially alter a third amino acid as well. The diversity of edits in the blastocysts with the majority being large deletions accurately forecasts the diversity of edits in fetuses, again with frequent large deletions observed.

CRISPR-Cas9技术为评估家畜物种中单个基因敲除后的生理反应提供了全新契机，拓展了我们对动物生物学的认知。在启动耗时冗长的胚胎移植以构建目标模式动物之前，优先测试单条向导序列（guides）的基因敲除效能是十分必要的。本实验旨在测试靶向猪受精卵中雄激素受体（androgen receptor, AR）的向导序列的敲除效能，并对由此产生的基因缺失进行表征。研究设计了两条靶向外显子2（Ensembl家猪参考基因组11.1，Ensembl Sus Scrofa 11.1）的向导序列。将上述向导序列通过电转染导入体外构建的猪受精卵后，将胚胎培养至囊胚期并进行DNA测序。经两条靶向外显子2的向导序列电转染后获得的囊胚中，超过80%存在基因缺失，且均为双等位基因缺失。尽管部分囊胚仅存在单条向导序列介导的编辑，但绝大多数囊胚存在由两条向导序列协同作用产生的大片段基因缺失。
实验方法
从商业屠宰场获取的猪卵巢的3-5mm卵泡中抽吸卵丘-卵母细胞复合体，经洗涤后于38.5℃、体外成熟培养40-44小时。将卵母细胞置于含约1000个经洗涤、部分获能精子的100μL液滴中，在空气环境含5%CO₂的条件下孵育5-6小时。受精后9小时，将受精卵在Opti-MEM™（Gibco）培养基中，使用CRISPR向导序列与Cas9蛋白进行电转染。将电转后的受精卵培养至囊胚期后，将单个囊胚置于10μL QE09050缓冲液（英国Biosearch Technologies公司）中裂解，经涡旋振荡、离心后，依次在65℃孵育6分钟、98℃孵育2分钟，随后于-17℃保存。通过两轮巢式PCR扩增DNA，将PCR产物在0.8%琼脂糖凝胶上进行电泳分离，切取目的条带后，使用QIAquick凝胶提取试剂盒（Qiagen #28704）提取DNA并送交测序。使用SnapGene®软件将测序序列与家猪参考基因组Sus Scrofa 11.1进行比对。选取9个代表性囊胚的两条CRISPR向导序列靶位点周围的序列，结果如图1所示。当使用靶向家猪AR基因外显子2的两条CRISPR向导序列组合进行电转染时（参考序列中靶位点以红色标注），绝大多数囊胚存在大片段基因缺失，仅1个囊胚存在6bp的缺失。该单一例编辑仅可能消除转录本中的2个氨基酸，或也可能改变第3个氨基酸。囊胚中编辑类型的多样性（以大片段缺失为主）可准确预测胎儿中的编辑多样性，后者同样频繁观察到大片段缺失。