UC Berkeley

It is projected that by the year 2050, global agriculture will need to increase the output of staple crops by 60% to feed the world’s rapidly growing population amidst threats posed by climate change and crop disease. One strategy to meet this challenge is to develop new genetic diversity in the germplasm to generate robust and resilient lines with beneficial traits. Conventional breeding has played an essential and major role in crop trait improvement. However, those methods can be difficult, laborious, and time-consuming. The development of effective technology for precision and time-saving plant breeding is required. A breakthrough tool to this end has been CRISPR-Cas9. Noteworthy for its simple sequence-specific programmability, the gene editing system has unleashed remarkable potential for plant biotechnology and functional genomics. Here, we explore several aspects and parameters to advance Cas9-mediated gene editing technology in the essential monocot food crops: rice and wheat. Early-stage plant gene-editing projects benefit greatly from a rapid transient pipeline using protoplasts to evaluate the efficacy of gene editing reagents. We describe here a novel and sustainable method for protoplast isolation from rice tissue and demonstrate their use in ribonucleoprotein (RNP) based Cas9 gene editing assays. Next, we focus on applications of Cas9 technology in wheat to engineer disease resistance. Wheat is a critical target organism because the complexity of its large allohexaploid genome has rendered genetic manipulation by classic methods extremely difficult. Using DNA plasmids encoding CRISPR-Cas9 gene editing reagents, we optimize conditions and demonstrate that all three homoeologous copies of genes in allohexaploid wheat can be simultaneously edited within a single generation. We recognize, however, that DNA plasmid gene editing approaches have a number of limitations including random integration into the plant genome and unpredictability of expression. To address this, we finally present significant improvements to DNA-free Cas9-RNP based gene editing in wheat. We show that increased temperature treatment greatly enhances Cas9-mediated editing efficiency and regenerate transgene-free edited wheat plants at a high rate. Lastly, we utilize this DNA-free gene editing method to generate de novo partial resistance to the agronomically important pathogen Parastagonospora nodorum.