Efficiency, Specificity and Temperature Sensitivity of Cas9 and Cas12a RNPs for DNA-free Genome Editing in Plants

• DOI: 10.3389/fgeed.2021.760820
• Publication Date: 2022-01-12T07:03:28Z
• Authors: Raviraj Banakar; Mollie Schubert; Gavin Kurgan; Krishan Mohan Rai; Sarah F. Beaudoin; Michael A. Collin...; Christopher A. Va...; Kan Wang; Feng Zhang
• Keywords: Genome Engineering; Protoplast

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target effects, mitigation random integration non-native DNA fragments, independence vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated is still at early development stage plants. In this study, we established an efficient simplified protoplast-based platform for RNP delivery, then evaluated efficiency, specificity, temperature sensitivity six Cas9 Cas12a proteins. Our results demonstrated that resulted frequencies (8.7–41.2%) various conditions, 22°C, 26°C, 37°C, with no significant sensitivity. LbCas12a often exhibited highest activities, while AsCas12a higher sequence specificity. The high activities RNPs 22° preferred by plant transformation tissue culture, led mutagenesis efficiencies (34.0–85.2%) protoplast-regenerated calli plants heritable mutants recovered next generation. This approach was further extended pennycress ( Thlaspi arvense ), soybean Glycine max ) Setaria viridis up 70.2% frequency. Together, study sheds light on choice achieve transgene-free