Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.)

Authors: PODDAR, SNIGDHA - University Of California; TANAKA, JACLYN - University Of California; RUNNING, KATHERINE - North Dakota State University; KARIYAWASAM, GAYAN - North Dakota State University; Faris, Justin; Friesen, Timothy; CHO, MYEONG-JE - University Of California; KATE, JAMIE - University Of California; STASKAWICZ, BRIAN - University Of California

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2022
Publication Date: 1/10/2023
Citation: Poddar, S., Tanaka, J., Running, K.L., Kariyawasam, G., Faris, J.D., Friesen, T.L., Cho, M., Kate, J.H., Staskawicz, B. 2023. Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.). Frontiers in Plant Science. 13. Article 1084700. https://doi.org/10.3389/fpls.2022.1084700.
DOI: https://doi.org/10.3389/fpls.2022.1084700

Interpretive Summary: The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, a technology known as CRISPR/Cas9 has been adopted widely for gene editing in plants. However, one undesirable result of conventional CRISPR/Cas9 technology is the random integration of ‘foreign’ DNA into the plant genome. Here, we describe to a method for gene editing in wheat using protein and RNA instead of DNA. The new method is highly efficient and results in no incorporation of unwanted DNA within the plant’s genome. Results from this work also led to the development of a pre-editing assay that can be used to greatly enhance efficiency of the gene editing process itself. This and other knowledge generated in this research resulted in a much-improved gene editing method for wheat in the absence of DNA introduction. To demonstrate the usefulness of the method, we produced edits to ‘turn off’ two disease susceptibility genes in wheat, which resulted in disease-resistant plants. The establishment of highly efficient, DNA-free gene editing technology holds promise for accelerated production of trait diversity in an expansive array of crops.

Technical Abstract: The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium tumefaciens or particle bombardment based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9 mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of two pathogenic effector susceptibility genes, resulting in insensitivity to corresponding necrotrophic effectors from Parastagonospora nodorum. The establishment of highly efficient, DNA-free gene editing technology holds promise for accelerated production of trait diversity in an expansive array of crops.