EAGER: Enabling Efficient Genome Editing for Advanced Biomanufacturing Applications

该项目解决了识别和纠正影响细胞培养系统效率的问题的挑战，这些问题用于生产大多数治疗性蛋白质。这种利用中国仓鼠卵巢（CHO）细胞的生物制造是价值1000亿美元产业的一部分。CHO细胞很难使用现代基因工程工具进行修饰，并且易于在其遗传物质（DNA）中发生随机变化。该项目将研究修复受损DNA的蛋白质，这是基因工程困难的原因，也是CHO细胞遗传不稳定性的原因，并有望对细胞系的生物学产生新的见解，并为这些细胞开发新的工具。学生将在跨学科的环境中接受培训，这将使他们接触到多种潜在的职业道路。该项目有望在中国仓鼠卵巢（CHO）细胞中实现更有效的基因组编辑；目前在这些细胞上进行合成生物学是有问题的，即使使用CRISPR和其他先进的方法。DNA双链断裂修复系统（执行基因组编辑所需的分子机器的一个组成部分）存在缺陷，从而导致合成生物学方法在这些细胞中的失败，这一假设将得到验证。DNA双链断裂修复系统的缺陷也可能是CHO细胞基因组不稳定的原因。该项目的目标是：1)根据序列相似性和功能优先顺序，系统地将CHO的DNA双链断裂修复基因替换为中国仓鼠的相应版本；2)每次替换后，定量评估该变化是否对基因组编辑效率和染色体稳定性产生了可测量的影响。

This project addresses the challenge of identifying and correcting problems that affect the efficiency of the cell culture system used to produce the majority of therapeutic proteins. Such biomanufacturing using Chinese hamster ovary (CHO) cells is part of a $100 billion industry. CHO cells are difficult to modify using modern genetic engineering tools and are prone to random changes in their genetic material (DNA). This project will examine proteins that repair damaged DNA as a cause of the difficulties in genetic engineering as well as the genetic instability of CHO cells, and promises to generate new insights into the biology of cell lines as well as developing new tools for these cells. Students will be trained in an interdisciplinary setting that will expose them to multiple potential career paths. This project is anticipated to result in more efficient genome editing in Chinese hamster ovary (CHO) cells; performing synthetic biology on these cells is currently problematic, even using CRISPR and other advanced approaches. The hypothesis will be tested that the DNA double strand break repair system (a component of the molecular machinery that is needed to perform genome editing) is flawed, thereby leading to the failure of synthetic biology approaches in these cells. Defects in the DNA double strand break repair system may also be the cause of the problematic genome instability in CHO cells. The goals of the project are 1) to systematically replace each of the DNA double strand break repair genes from CHO with the corresponding version from the Chinese hamster following a priority list based on sequence similarity and function; and 2) after each replacement, to quantitatively evaluate whether the change has had a measureable impact on efficiency of genome editing and on chromosomal stability.