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.
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