Developing a CRISPR toolbox for efficient gene editing in crops

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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 The wild-type MAD7nuclease has ~2-3% efficiency of target DNA cleavage at 27C in plant cells (unpublished data,Phytoform Labs).