2019 ASHS Annual Conference

Genome editing enables precise manipulation of specific genomic sites with the newly developed CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system being the most promising genome editing tool. DNA-based delivery of the CRISPR-Cas9 system has been routinely used in various plant species. However, protein delivery of the in vitro-translated Cas9/gRNA ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has advantages such as ease of use, low cost, relative ease to be adapted to high throughput systems, and lower off-target cleavage rates. The present study is the first time to successfully develope lipofection-mediated transfection as a novel approach for protein delivery of the preassembled Cas9/gRNA RNP complex into plant cells for genome editing. This approach was achieved using Lipofectamine 3000- or RNAiMAX-mediated transfection. Using either method, we demonstrated that the pre-complexed Cas9/gRNA RNP complex in which a GFP was tagged in-frame to the 3’-end of the Cas9 protein was successfully delivered into non-transgenic tobacco cv. Bright Yellow-2 (BY-2) protoplasts. The optimal lipofectamine 3000- and RNAiMAX-mediated protein delivery efficiencies were 66 and 48%, respectively. We also developed a new biolistics approach based on the published proteolistics method. When delivered into transgenic BY-2 protoplasts or cells with 60% cells overexpressing an orange fluorescent protein (OFP) reporter gene pporRFP with each gRNA targeting the pporRFP gene, we demonstrated that Lipofectamine 3000-mediated protein delivery had higher targeted mutagenesis frequency than the PEG-mediated DNA delivery method and the biolistics-mediated protein delivery method. Thus, the newly developed lipofection approach is sufficiently robust for the use of the DNA-free Cas9/gRNA RNP technology for genome editing in plant cells.