EAGER: Biomanufacturing: CRISPR to increase the homogeneity and efficiency of stem cell differentiation

干细胞在衰老、受伤和患病的组织和器官的再生方面是有希望的，但为了实现它们的治疗潜力，不需要的细胞类型的生长应该被阻止，并提高将它们转化为特定细胞类型的效率是必要的。这项拟议的工作将研究一种基因组编辑方法，以消除不需要的细胞系的生长。本项目的总体目标是通过CRISPR减少脱靶细胞类型，提高干细胞分化的效率和特异性。CRISPR/Cas9介导的敲除参与血清素能、谷氨酸能和gaba能表型成熟的转录因子。将对克隆进行DNA测序，以确认涉及血清素、谷氨酸和GABA合成途径的区域是否缺失，而那些缺乏这些基因的克隆将用FI （forskolin和IBMX）处理，以诱导更高比例的细胞表达多巴胺能表型。该方法还可用于提高神经干细胞分化的效率和特异性。预计结果将很容易转移到诱导多能干细胞、胚胎干细胞和成体干细胞。从教育的角度来看，这个项目将为学生提供跨学科的培训，让他们有机会整合不同领域的知识来研究非传统的工程问题。研究人员将招募并留住女性和少数族裔工程专业的学生参与研究项目。

PI: Chan, Christina Proposal Number: 1547518Stem cells are promising for the regeneration of aged, injured and diseased tissues and organs, but to realize their therapeutic potential growth of unwanted cell types should be arrested, and improvement in efficiency of converting them to a specific cell type is necessary. The proposed work will examine a genome editing approach for eliminating growth of unwanted cell lineages. The overall goal of this project is to increase the efficiency and specificity of stem cell differentiation by reducing off-target cell types through CRISPR. CRISPR/Cas9 mediated knockout of transcription factors involved in the maturation of serotonergic, glutamatergic and GABAergic phenotypes. Clones will be DNA sequenced to confirm deletions in the regions involved in serotonin, glutamate and GABA synthesis pathways, and those lacking these genes will be treated with FI (forskolin and IBMX) to induce a higher percentage of cells to express a dopaminergic phenotype. This method will also be applied to increase the efficiency and specificity of neural stem cell differentiation. The results are anticipated to easily transfer to induced pluripotent, embryonic, and adult stem cells. From an educational standpoint, this project will provide interdisciplinary training to students with an opportunity to integrate knowledge from different fields to investigate non-traditional engineering problems. The investigators will recruit and retain female and minority engineering students into the research project.