Genome Engineering and iPSC Center

基因组工程和iPSC中心（GEiC）共享资源：项目摘要 (iPSC：诱导多能干细胞） 过去五年见证了CRISPR基因组操作技术的非凡发展。 基因组工程和iPSC中心（GEiC）共享资源（SR）随着技术的成熟而增长。在 上一次更新，GEiC是一个新的SR，在建立CRISPR的早期阶段有五个FTE， 工作流程。今天，我们拥有14.5名全职员工，提供广泛的服务，以满足各种需求和级别 Siteman癌症中心（SCC）成员在细胞（癌症和iPS细胞）和动物模型上的研究。过去 五年来，GEiC已经完成了500多个各种癌细胞系的工程项目， 在iPSC和hESC中，为超过350种小鼠模型设计并验证了试剂，验证了3500种gRNA， 储存了超过400个患者样本，并重新编程了超过110个来自患者的iPSC。 推出的新服务包括：1）全血（包括凝血）的银行和重编程， 允许患者样品提交的极大灵活性，2）将iPSC分化成各种组织类型， 干细胞、外周神经元细胞、造血干细胞、CD 34+细胞和巨噬细胞，3）文库 用于CRISPR筛选和数据分析的构建，4）用于CRISPR介导的基因活化的试剂， 失活和使用碱基编辑器引入SNP而不切割染色体，5）接着 用于细胞系认证的基于测序的短串联重复序列（STR）分析。另外我们 在细胞和动物模型创建方面进行了重大工艺改进，例如优化单细胞克隆 在iPSC的基因编辑过程中的效率，通过使用 具有末端受保护的单链寡DNA供体的CRISPR核糖核蛋白复合物和电穿孔 单细胞小鼠胚胎，而不是显微注射，这使我们能够实现超过95%的成功率， 在小鼠中获得挑战性条件等位基因（floxing）。 在下一个项目期间，我们将在我们成功的基础上，努力使疾病建模更加相关 并向SCC成员开放。我们将扩大可以与iPSC区分的组织类型， 建立iPSC的同时重编程和编辑的方案，用于更快的疾病/纠正 等基因对的产生和可逆的永生化以增殖原代细胞类型而不失去它们的 生理特性我们还在努力建立一个健康的iPSC控制库， 不同的种族背景，并进一步提高细胞和胚胎的基因组工程效率， 自动化和流程改进。与此同时，我们将继续在以下领域取得进展： 基因组工程和iPSC，并确保SCC成员提供最先进的技术。

GENOME ENGINEERING AND iPSC CENTER (GEiC) SHARED RESOURCE: PROJECT SUMMARY (iPSC: induced pluripotent stem cells) The past five years witnessed the extraordinary development of CRISPR technology for genome manipulation. The Genome Engineering and iPSC Center (GEiC) shared resource (SR) grows as the technology matures. At last renewal, the GEiC was a new SR with five FTEs at a fairly early stage of establishing CRISPR-involving workflows. Today we have 14.5 FTEs with a wide range of service offerings to meet the various needs and levels of Siteman Cancer Center (SCC) members on cell (both cancer and iPS cells) and animal models. In the past five years, the GEiC has completed over 500 engineering projects in various cancer cell lines and another 200 in iPSC and hESCs, designed and validated reagents for over 350 mouse models, validated 3500 gRNAs, banked over 400 patient samples, and reprogrammed over 110 lines of patient-derived iPSCs. New services launched include 1) banking and reprogramming from whole blood, including clotted blood, allowing great flexibility for patient sample submission, 2) differentiation of iPSCs into various tissue types, neural stem cells, peripheral neuronal cells, hematopoietic stem cells, CD34+ cells, and macrophages, 3) library construction for CRISPR screens and data analysis, 4) reagents for CRISPR-mediated gene activation and inactivation and the use of base editor for introducing SNPs without cleaving the chromosomes, 5) next generation sequencing-based short tandem repeat (STR) profiling for cell line authentication. Additionally, we made significant process improvement in cell and animal model creation, such as optimized single cell cloning efficiency during gene editing of iPSCs, increased efficiency and consistency of SNP introduction by using CRISPR ribonucleoprotein complex with end protected single stranded oligo DNA donors, and electroporation of single-cell mouse embryos instead of microinjection, which allows us to achieve over 95% success rate on obtaining the challenging conditional alleles (floxing) in mice. In the next project period, we will build on our success and strive to make disease modeling more relevant and accessible to SCC members. We will expand the tissue types that can be differentiated from iPSCs and establish protocols for simultaneous reprogramming and editing of iPSCs for speedier disease/corrected isogenic pair creation and for reversible immortalization to proliferate primary cell types without losing their physiological characteristics. We are also working on establishing a repository for healthy iPSC controls of various ethnic backgrounds and further improving genome engineering efficiency in cells and embryos through automation and process improvement. In the meantime, we will keep up with advancement in the fields of genome engineering and iPSCs and ensure SCC members are served with the most up-to-date technologies.