CRISPR DNA- and RNP-Mediated Genome Editing via Nicotiana benthamiana Protoplast Transformation and Regeneration.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated system) has become the multipurpose tool to manipulate plant genome via their programmable sequence recognition, binding, and cleavage activities. Efficient plant genome modification often requires robust plant transformation. For most plant species, the CRISPR/Cas reagents are delivered into plants as plasmids by Agrobacterium-mediated T-DNA transfer or biolistic approaches. However, these methods are generally inefficient, heavily genotype dependent, and low throughput. Among the alternative plant transformation approaches, the protoplast-based transformation holds the potential to directly deliver DNA, RNA, or protein molecules into plant cells in an efficient and high-throughput manner. Here, we presented a robust and simplified protocol for protoplast-based DNA/ribonucleoprotein (RNP )-mediated genome editing in the model species Nicotiana benthamiana. Using this protocol, we have achieved the gene editing efficiency at 30-60% in protoplasts and 50-80% in regenerated calli and plants. The edited protoplasts can be readily regenerated without selection agents owing to highly efficient DNA or preassembled RNP transformation frequency. Lastly, this protocol utilized an improved culture media regime to overcome the complex media composition used in the previous studies. It offers quick turnaround time and higher throughput to facilitate the development of new genetic engineering technologies and holds the promise to combine with other genetic and genomic tools for fundamental and translational plant research.