Improving CRISPR workflows in plants

Full project Title

Improving CRISPR workflows in crop species for increased genome editing efficiency.

Project summary

The greatest bottleneck for widespread adoption of genome editing technologies to improve crop varieties is the incredibly laborious and time-consuming process required to generate a gene-edited plant. Accordingly, it is imperative to be confident about the efficiency of the gene editing approach and selected gRNAs before embarking on such a time-consuming project. However, the plant community is currently lacking 1) an efficient and accessible method for in planta gRNA evaluation and 2) a comprehensive assessment of published methods for quantifying CRISPR edits in plants that can be used routinely and importantly, standardized across research groups. This project will address both of these roadblocks to widespread adoption of this technology in plants.

Initially, we will perform a comparison of different methods used to quantify CRISPR edits in plants, assessing the advantages and disadvantages of these methods and identifying optimal methods based on several important criteria. Subsequently, we will use this knowledge to develop a rapid workflow allowing research groups to evaluate the genome editing efficiency of the selected CRISPR gRNAs for plant applications. This workflow will allow ranking of different gRNAs based on efficiency in plants before investing months or years needed for transformation and regeneration. Furthermore, we will expand the versatility of this workflow to assess gRNA performance for individual alleles or between highly similar sequences to overcome issues with increased ploidy and gene duplication events.

Potential Outcomes

We expect that the outcomes of this project will provide standards and guidelines for plant scientists when evaluating CRISPR editing efficiencies for plant genome editing, especially when CRISPR edits are anticipated to be heterogenous. Recommendations developed as an outcome of this study will form a point of reference for future plant genome editing projects throughout UQ.

In addition, the workflow developed will allow plant scientists to rapidly select the best performing gRNA sequence for their application of CRISPR technologies in plant basic research, biotechnology or crop improvements.