CRISPR Screens

While CRISPR systems are generally effective, there can be a wide range of gene-editing efficiencies, depending on the plant species and/or method of delivery. For example, our experience is that base editors are unpredictable, and most target sites are non-functional. To increase gene editing frequencies and improve the recovery of useful, gene-edited plants, the group is focused on the entire gene-editing workflow, from optimizing protocols to vector components and downstream analysis and isolation of mutants of interest.

CRISPR screens have become a routine tool in many areas of biological research but have been largely undeveloped in the plant community. The Plant Genome Editing (PGE) group, in collaboration with a number of other PSB groups and collaborators at the Flemish research institute ILVO, is developing a number of protocols and technologies to enable CRISPR screens in plant research. The PGE lab is currently performing CRISPR screens on moss and Arabidopsis.

The PGE lab has developed a software tool, SMAP design, that can generate gRNA and amplicon designs automatically on hundreds of genes in a few minutes to lower the barrier of starting a CRISPR screen. To keep the CRISPR screening population to a minimum, high-efficiency CRISPR vectors are needed. We compared different CRISPR architectures (promoters to express Cas9 and different NLS combinations to get Cas9 into the nucleus) and were able to find an architecture that was more than 3-fold more efficient than standard architectures. The PGE lab is currently developing cloning tools to assemble a large sets of gRNAs and tools to create large deletions in moss.