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

我们正在猪身上建立人类癌症模型，以帮助开发新的诊断程序和治疗方法。CRISPR/Cas9基因编辑极大地简化了大型哺乳动物的基因失活。最近报道了一种基于表达Cas9的转基因小鼠的特别有效的策略。通过引入靶向感兴趣基因的引导rna，可以在选定的体细胞中实现基因失活。这种体细胞工程有许多用途，包括模拟导致特定癌症的自发突变。我们建议将这一技术推广到猪身上，产生Cas9转基因猪品系。这将大大增加可以研究的基因数量，减少所需时间，从而大大增强癌症建模的能力和范围。重要的是，它将根据替换、减少和改进实验动物的原则，减少所需动物的数量。这项工作利用了我们已经培育出的三种转基因猪系。我们先前确定了猪ROSA26位点，并利用它放置了一个无处不在表达的Cre重组双荧光报告基因。我们还培育了具有关键癌症相关基因基因靶向突变的猪，包括引发结直肠息肉病的APC1311突变（与人类APC1309同源），以及可诱导的致癌KRASG12D（结直肠和其他癌症的重要驱动因素）。我们将培育组成型和可诱导型Cas9转基因，将它们通过靶向ROSA26的基因放置，并通过核移植制造猪。使用的细胞将携带APC1311突变，因此创始动物将患上息肉。原理验证研究将集中在本构Cas9上。靶向癌症相关基因的引导rna将在内镜下引入远端肠道息肉。后续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引入同样携带荧光Cre报告基因的APC1311动物的息肉中，确定将核酸递送到体内息肉的最佳方法（例如电穿孔，AAV载体）。活检样本荧光显微镜显示的cre重组细胞比例将提供转染/转导方法成功的衡量标准。如果时间允许，我们将使用VIL1（绒毛蛋白）启动子，在体内测试cre诱导的Cas9的局部和细胞类型特异性激活。我们未来的计划是将crer诱导的Cas9与crer诱导的KRASG12D结合起来，研究致癌KRAS表达与其他癌症相关基因失活的关系。这将包括KRAS效应物的选择性失活，以分析结直肠癌的重要信号通路。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应用于各种领域。

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.