ROSA26-Cas9 transgenic pigs: a tool for in vivo genome editing

我们正在猪身上模拟人类癌症，以帮助开发新的诊断程序和治疗方法。CRISPR/Cas9基因编辑大大简化了大型哺乳动物的基因失活。最近有报道称，一种特别有效的策略是基于表达Cas9的转基因小鼠。这可以通过引入针对感兴趣基因的引导RNA，在选定的体细胞中使基因失活。这种体细胞工程有很多用途，包括模仿导致特定癌症的自发突变。我们建议通过产生Cas9转基因猪品系来将这项技术扩展到猪。这将大大增加可供研究的基因数量，并减少所需的时间，从而显著增强癌症建模的能力和范围。重要的是，根据替换、减少和改进实验动物的原则，它将减少所需的动物数量。这项拟议的工作利用了我们已经培育的三个转基因猪品系。我们以前发现了猪rosa26基因，并用它定位了一个普遍表达的Cre重组的双荧光报告基因。我们还产生了关键癌症相关基因的基因靶向突变，包括引发大肠息肉的APC1311突变(与人类APC1309同源)，以及CRE诱导形式的致癌KrasG12D，KrasG12D是结直肠癌和其他癌症的重要驱动因素。我们将产生结构性和Cre诱导的Cas9转基因基因，通过基因靶向将它们置于rosa26上，并通过核移植使猪成为猪。使用的细胞将携带APC1311突变，因此创始人动物将患上息肉。原则证明研究将侧重于构成CAS9。针对癌症相关基因的引导RNA将通过内窥镜被引入远端肠道的息肉中。后续的结肠镜检查将监测表型并收集样本进行分子和免疫组织学分析。在产生创始动物的同时，我们将通过将Cre引入APC1311动物的息肉中来确定将核酸运送到体内的最佳方法(例如电穿孔、AAV载体)，APC1311动物也携带Cre荧光报告。活检样本的荧光显微镜显示的Cre重组细胞的比例将提供转基因/转导方法成功与否的衡量标准。如果时间允许，我们将在体内测试Cre诱导的Cas9的局部和细胞类型的特异性激活，例如使用VIL1(Villin)启动子。我们未来的计划是将Cre诱导的Cas9和Cre诱导的KrasG12D结合起来，研究致癌KRAS的表达与其他癌症相关基因的失活。这将包括选择性地灭活KRAS效应器，以分析对结直肠癌重要的信号通路。Cas9猪将成为许多研究人员的有用资源，使猪的基因组工程能够在广泛的领域应用。

We are modelling human cancers in pigs to aid development of new diagnostic procedures and treatments. CRISPR/Cas9 gene editing has dramatically simplified gene inactivation in large mammals. A particularly powerful strategy has recently been reported based on transgenic mice that express Cas9. This enables gene inactivation in chosen somatic cells by introducing guide RNAs targeted to genes of interest. Such somatic cell engineering has many uses, including mimicking spontaneous mutations responsible for particular cancers. We propose extending this technology to pigs by generating Cas9 transgenic pig lines. These would significantly enhance the power and scope of cancer modelling by greatly increasing the number of genes that can be investigated and reducing the time necessary. Importantly it would reduce the number of animals needed, in accordance with the principle of replacement, reduction and refinement for work with experimental animals. The work proposed makes use of three genetically modified pig lines we have already generated. We previously identified the porcine ROSA26 locus and used it to place a ubiquitously expressed dual fluorescent reporter of Cre recombination. We have also generated pigs with gene-targeted mutations in key cancer-related genes, including the APC1311 mutation (orthologous to human APC1309), which initiates colorectal polyposis, and a Cre-inducible form of oncogenic KRASG12D an important driver of colorectal and other cancersWe will generate constitutive and Cre-inducible Cas9 transgenes, place them by gene targeting at ROSA26 and make pigs by nuclear transfer. The cells used will carry the APC1311 mutation, so founder animals will develop polyps. Proof-of-principle studies will focus on constitutive Cas9. Guide RNAs targeted to cancer-related genes will be introduced endoscopically into polyps in the distal gut. Follow-up colonoscopies will monitor the phenotype and collect samples for molecular and immunohistological analyses.While founder animals are being generated we will determine the best method of delivering nucleic acids into polyps in vivo (e.g. electroporation, AAV vectors) by introducing Cre into polyps in APC1311 animals that also carry the fluorescent Cre reporter. The proportion of Cre-recombined cells visualised by fluorescence microscopy of biopsy samples will provide a measure of the success of transfection/transduction methods. If time allows, we will test local and cell-type specific activation of Cre-inducible Cas9 in vivo, using for example the VIL1 (villin) promoter. Our future plans are to combine Cre-inducible Cas9 with Cre-inducible KRASG12D to investigate oncogenic KRAS expression with inactivation of other cancer-related genes. This will include selective inactivation of KRAS effectors to analyse signalling pathways important for colorectal cancer. The Cas9 pigs will be a useful resource for many researchers, enabling genome engineering in pigs for a wide variety of fields.