Developing a CRISPR toolbox for efficient gene editing in crops

该项目旨在利用增强的分子和计算工具为农作物开发更好的基因编辑工具。我们将在大肠杆菌中构建这些原型，作为快速实施设计-构建-测试-学习（DBTL）周期的更可行的测试平台。由于实验系统的复杂性（基因组大小、多倍体）和用于进行修饰的工具的分子特性，在植物中进行有效的基因编辑仍然是一个挑战。我们将使用定向进化和机器学习来改进核酸酶，重点关注蛋白质的稳定性、活性、大小及其在宿主细胞中的基因表达负担。该项目将专注于MAD7 （ErCas12a）核酸酶，这是一种crispr相关蛋白，可用于多种生物的基因编辑。它比其他替代品具有实际优势，因为它可以用于商业产品开发而不产生版税。然而，与标准核酸酶如Cas9和Cpf1 （LbCas12a）相比，MAD7在室温下的稳定性和活性使其难以工作。对于非转基因植物基因编辑方案尤其如此，其中核酸酶作为纯化的核糖核蛋白提供，以提高效率并减少脱靶效应。野生型mad7核酸酶在植物细胞27℃条件下的靶DNA切割效率约为2-3%（未发表的数据，Phytoform Labs）。

This project aims to develop better gene editing tools for crop plants using enhanced molecularand computational tools. We will prototype these in E. coli as a more feasible test platform forrapid implementation of the design-build-test-learn (DBTL) cycle.Efficient gene editing in plants remains a challenge, due to the complexity of the experimentalsystem (genome size, polyploidy) and the molecular properties of the tools used to make themodifications. We will use directed evolution and machine learning to improve the nucleases witha focus on stability, activity, size of the protein, and its gene expression burden in host cells.The project will focus on MAD7 (ErCas12a) nuclease, a CRISPR-associated protein that can be usedfor gene editing in a wide variety of organisms. It has practical advantages over alternativesbecause it can be used for commercial product development without incurring royalties.However, the stability and activity of MAD7 at room temperature make it difficult to work withcompared to standard nucleases such as Cas9 and Cpf1 (LbCas12a). This is particularly true fornon-transgenic plant gene editing protocols where the nuclease is supplied as a purifiedribonucleoprotein to improve the efficiency and reduce off-target effects. The wild-type MAD7nuclease has ~2-3% efficiency of target DNA cleavage at 27C in plant cells (unpublished data,Phytoform Labs).