Improving CRISPR-Cas9 mediated gene editing in plants

Naim F1, Shand K1, Roden S1, Hayashi S1, O'Brien M2, Johnson A2, Dugdale B1 and Waterhouse P1

  1. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia.
  2. School of BioSciences, The University of Melbourne, Melbourne, Australia.

CRISPR-Cas9 driven gene editing of crops is rapidly advancing agricultural biotechnology. The process relies on DNA double stranded breaks at user defined genomic loci and repair by non-homologous end joining and/or homologous recombination. However, there are limitations in achieving precise and efficient editing of genes. These limitations include optimum design rules for gRNAs and delivery of Cas9 editing cassette. There are many gRNA design rules and online algorithms available to design gRNAs targeting genes in humans. We used a number of these tools to design and test the efficiency of gRNAs in Nicotiana benthamiana, banana, rice and tomato. Our results showed that there is no consensus between predictions by these algorithms and efficiency of obtaining mutations in plants. We discovered that targeting a single gene with two gRNAs increased the frequency of gene edits. To further improve editing, we also explored delivery of Cas9 plasmid using various DNA viruses. Here we report our exploration of the techniques and progress in efficiently editing plant genes.