Efficiency, specificity and temperature sensitivity of Cas9 and Cas12a RNPs for DNA-free gene editing in plants

Gene editing via the CRISPR/Cas ribonucleoproteins (RNPs) offers several advantages over the plasmid DNA-based methods, including no vector constructions, no random integration of non-native DNA fragments, less off-target effects, and potentially less regulatory restrictions. Compared to their use in animal systems, RNP-mediated gene editing is still at the early development stage in plants. In this study, after establishing an efficient and simplified protoplast-based gene editing platform for the CRISPR/Cas RNP delivery, we evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that the Cas9 and Cas12a RNPs can induce efficient mutations at various temperature conditions, from 22 C, 26 C to 37 C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of the CRISPR/Cas RNPs at 22 C and 26 C , the temperature preferred by plant transformation and tissue culture, led to the high mutagenesis efficiencies (34.0% to 85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to other dicot and monocot plants with up to 70.2% mutagenesis frequency observed in the model plant, Setaria viridis. Together, this study sheds light on the choice of RNP reagents for plant gene editing. It demonstrates that RNP-mediated protoplast gene editing is a promising technique for rapidly optimizing the CRISPR/Cas systems as well as an effective platform for generating transgene-free gene edited plants and performing CRISPR screening in plant cells.