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

使用 Cas9-Bxb1 整合酶工具箱将大型 (10-100 kb) DNA 构建体定点整合到小鼠基因组和人类诱导多能干细胞中

• 批准号: 10654838
• 负责人: Vishnu Hosur
• 金额: $ 63.34万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654838
• 关键词: Address; Animals; Bacteria; Breast Cancer Model; CRISPR/Cas technology; Cancer Model; Cell Line; Chromosomes; Code; Collaborations; Communities; Congenital Megacolon; Couples; Cre lox recombination system; DNA; DNA Integration; DNA Sequence; Development; Disease; Disease Progression; Disease model; Electroporation; Engineering; Enhancers; Excision; Fluorescence Microscopy; Future; Gene Rearrangement; Gene Transfer Techniques; Generations; Genes; Genetic Engineering; Genetic Recombination; Genetic Transcription; Genome; Genomics; Goals; Guide RNA; Human; Integrase; Left; Length; Link; Literature; Malignant Neoplasms; Mediating; Microinjections; Modeling; Mus; Organism; Play; Protocols documentation; Public Health; Reaction; Reporter; Research; Research Personnel; Resource Development; Role; Serine; Site; System; Techniques; Technology; Testing; Toxic effect; Transgenic Mice; Transposase; Untranslated RNA; Variant; Viral; Work; arm; blastocyst; digital; experimental study; genetic makeup; genetic variant; genome editing; human disease; human embryonic stem cell; human model; human stem cells; improved; induced pluripotent stem cell; innovation; mouse development; mouse genome; mouse model; multidisciplinary; nanopore; novel; prevent; recombinase; repaired; risk variant; site-specific integration; stem cell model; success; tool; tool development; transmission process; triple-negative invasive breast carcinoma; zygote

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.

项目总结/摘要 该项目的长期目标是提高研究人员创造忠实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系统彻底改变了基因组编辑，但缺乏有效整合 小鼠受精卵和小鼠及人干细胞中的大DNA构建体（> 10个内切酶; kb）。这种限制 严重阻碍了人类疾病，包括癌症的建模。例如，串联重复（TD）， 超级增强子（SE;转录增强子的大簇）和大的非编码结构变体具有 与人类疾病有关，包括癌症，但现有技术不允许建立如此大的模型 在整个动物或细胞系中的变体。为了填补这一空白，我们将开发一个基因编辑工具箱， CRISPR-Cas9系统的精确性与丝氨酸整合酶Bxb 1的保真度和效率， 在小鼠、小鼠胚胎干细胞和人类诱导的细胞中快速、有效地插入大DNA构建体， 多能干细胞（hiPSC）。Bxb 1整合酶使用DNA附着位点（基因组中的attP， 供体DNA）作为催化有效转基因的底物。我们展示了我们创新的Cas9-Bxb 1工具箱 可以在小鼠中精确整合长度高达~43 kb的DNA构建体。在这里，我们将在三个目标中进一步发展 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重组的过程。目的1：优化Cas9-Bxb 1工具箱以插入大DNA（10至1000个）。 100 kb）构建到小鼠受精卵中。我们将使用不同长度的报告构建体来确定 最大长度的DNA构建体，可以有效地插入，并将验证一个一步方案，快速 产生转基因小鼠，而不需要首先产生和表征具有附着位点的小鼠。 目标2：使用Cas9-Bxb 1工具箱生成人类疾病（包括癌症）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 23.7-kb TD的hiPSC模型）、三阴性乳腺癌的hiPSC模型（27.2-kb SE）和先天性巨结肠的小鼠模型 疾病（~80-kb人类风险等位基因）。目标3：使用Cas9-Bxb 1工具箱生成DNA 重新安排在cre-lox重组系统中，cre催化loxP序列两侧的两个loxP位点之间的重组， 一个靶位点，使不同的DNA重排。研究表明，cre-重组功效有限 通过IoxP位点间距离和靶向的特定基因组位点。我们将确定Cas9- Bxb 1工具箱比cre-lox更有效地产生DNA重排，通过确定Bxb 1介导的 在不同的attP/attB间距离的重组效率。该项目的成功完成将提供 社区为未来的研究提供了三种新的模型，以及一种用于开发新的和改进的 癌症和其他疾病的小鼠和hiPSC模型。