Site-specific Integration of Large (10-100 kb) DNA Constructs into the Mouse Genome and Human Induced Pluripotent Stem Cells Using the Cas9-Bxb1 Integrase Toolbox

项目摘要/摘要 这个项目的长期目标是提高研究人员创造忠诚的小鼠和干细胞的能力 人类癌症和其他疾病的模型。目前可用的基因工程方法，包括 CRISPR-Cas9系统为基因组编辑带来了革命性的变化，但缺乏有效整合 小鼠受精卵、小鼠和人类干细胞中的大DNA结构(&gt；10kb；kb)。这一限制 显著阻碍了对包括癌症在内的人类疾病的建模。例如串联复制(TDS)， 超级增强子(SE；转录增强子的大簇)和大型非编码结构变体 与包括癌症在内的人类疾病有关，但现有技术不允许对如此大的 在整个动物或细胞系中的变异。为了填补这一空白，我们将开发一个基因编辑工具箱，将 CRISPR-Cas9系统的精密度与丝氨酸整合酶Bxb1的保真度和效率 在小鼠、小鼠胚胎干细胞和人诱导的大DNA构建物中快速、高效地插入 多能干细胞(HiPSC)。Bxb1整合酶使用DNA连接位点(基因组中的attP，基因组中的attB 供体DNA)作为催化高效转基因的底物。我们展示了我们创新的Cas9-Bxb1工具箱 可以在小鼠体内精确整合长度高达~43kb的DNA构建物。在这里，我们将在三个目标上进一步发展 并验证工具箱以实现大DNA构建体(~100 kb)的精确转基因并促进 DNA重排的产生。目标1：优化Cas9-Bxb1工具箱以插入大DNA(10到 100kb)构建成小鼠受精卵。我们将使用不同长度的报告器构造来确定 可有效插入的DNA构建的最大长度，并将验证快速一步协议 无需首先生成带有附着位置的转基因小鼠并确定其特征，即可生成转基因小鼠。 目的2：使用Cas9-Bxb1工具箱建立包括癌症在内的人类疾病的小鼠和HiPSC模型。 使用我们的工具箱插入大的基因组变异，我们将产生乳腺癌的小鼠模型(插入 23.7kb的TD)、三阴性乳腺癌的HiPSC模型(27.2kb SE)和先天性巨结肠症的小鼠模型 疾病(约80kb的人类风险等位基因)。目标3：能够使用Cas9-Bxb1工具箱生成DNA 重新安排。在cre-lox重组系统中，cre催化两个loxP位点之间的重组。 一个靶点，使多种DNA重排成为可能。研究表明重组的效果是有限的。 根据loxP位点之间的距离和目标特定的基因组位点。我们将确定Cas9是否- 通过确定Bxb1介导的DNA重排，Bxb1工具箱比cre-lox更有效地产生DNA重排 不同attP/attB距离下的重组效率。该项目的成功完成将为 社区为未来的研究提供了三个新的模型，并为开发新的和改进的工具提供了一个通用的工具 小鼠和HiPSC癌症和其他疾病的模型。

PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to increase the ability of researchers to create faithful mouse and stem-cell models of human cancers and other diseases. Currently available genetic-engineering approaches, including the CRISPR-Cas9 system, which has revolutionized genome editing, lack the capacity for efficient integration of large DNA constructs (> 10 kilobases; kb) in mouse zygotes and mouse and human stem cells. This limitation significantly hinders the modeling of human diseases, including cancer. For example, tandem duplications (TDs), super-enhancers (SEs; large clusters of transcriptional enhancers), and large non-coding structural variants have been linked to human diseases, including cancers, but available technologies do not permit modeling such large variants in whole animals or cell lines. To fill this gap, we will develop a gene-editing toolbox that couples the precision of the CRISPR-Cas9 system with the fidelity and efficiency of the serine integrase Bxb1 to enable rapid, efficient insertion of large DNA constructs in mice, mouse embryonic stem cells, and human induced pluripotent stem cells (hiPSCs). Bxb1 integrase uses DNA attachment sites (attP in the genome, attB in the donor DNA) as substrates for catalyzing efficient transgenesis. We show that our innovative Cas9-Bxb1 toolbox can precisely integrate DNA constructs up to ~43 kb in length in mice. Here, in three aims we will further develop and validate the toolbox to enable precise transgenesis of large DNA constructs (~100 kb) and to facilitate generation of DNA rearrangements. Aim 1: Optimize the Cas9-Bxb1 toolbox for insertion of large DNA (10 to 100 kb) constructs into mouse zygotes. We will use reporter constructs with differing lengths to determine the maximum length of DNA construct that can be inserted efficiently, and will validate a one-step protocol for rapid generation of transgenic mice without the need to first generate and characterize mice with attachment sites. Aim 2: Generate mouse and hiPSC models of human diseases, including cancer, using the Cas9-Bxb1 toolbox. Using our toolbox to insert large genomic variants, we will generate a mouse model of breast cancer (insertion of a 23.7-kb TD), hiPSC model of triple negative breast cancer (27.2-kb SE), and mouse model of Hirschsprung disease (~80-kb human risk allele). Aim 3: Enable use of the Cas9-Bxb1 toolbox for generation of DNA rearrangements. In cre-lox recombination systems, cre catalyzes recombination between two loxP sites flanking a target locus, enabling diverse DNA rearrangements. Studies suggest that cre-recombination efficacy is limited by the inter-loxP-site distance and the particular genomic site targeted. We will determine whether the Cas9- Bxb1 toolbox is more efficient than cre-lox for generation of DNA rearrangements, by determining Bxb1-mediated recombination efficacy at different inter-attP/attB distances. Successful completion of this project will provide the community with three new models for future studies, and a versatile tool for development of novel and improved mouse and hiPSC models of cancer and other diseases.