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

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

Temperature sensitivity of termites determines global wood decay rates

Animals, such as termites, have largely been overlooked as global-scale drivers of biogeochemical cycles1,2, despite site-specific findings3,4. Deadwood turnover, an important component of the carbon cycle, is driven by multiple decay agents. Studies have focused on temperate systems5,6, where microbes dominate decay7. Microbial decay is sensitive to temperature, typically doubling per 10°C increase (decay effective Q10 = ~2)8–10. Termites are important decayers in tropical systems3,11–13 and differ from microbes in their population dynamics, dispersal, and substrate discovery14–16, meaning their climate sensitivities also differ. Using a network of 133 sites spanning 6 continents, we report the first global field-based quantification of temperature and precipitation sensitivities for termites and microbes, providing novel understandings of their response to changing climates. Temperature sensitivity of microbial decay was within previous estimates. Termite discovery and consumption were both much more sensitive to temperature (decay effective Q10 = 6.53), leading to striking differences in deadwood turnover in areas with and without termites. Termite impacts were greatest in tropical seasonal forests and savannas and subtropical deserts. With tropicalization17 (i.e., warming shifts to a tropical climate), the termite contribution to global wood decay will increase as more of the earth’s surface becomes accessible to termites.

Temperature sensitivity of Notch signaling underlies species-specific developmental plasticity and robustness in amniote brains

Ambient temperature significantly affects developmental timing in animals. The temperature sensitivity of embryogenesis is generally believed to be a consequence of the thermal dependency of cellular metabolism. However, the adaptive molecular mechanisms that respond to variations in temperature remain unclear. Here, we report species-specific thermal sensitivity of Notch signaling in the developing amniote brain. Transient hypothermic conditions increase canonical Notch activity and reduce neurogenesis in chick neural progenitors. Increased biosynthesis of phosphatidylethanolamine, a major glycerophospholipid components of the plasma membrane, mediates hypothermia-induced Notch activation. Furthermore, the species-specific thermal dependency of Notch signaling is associated with developmental robustness to altered Notch signaling. Our results reveal unique regulatory mechanisms for temperature-dependent neurogenic potentials that underlie developmental and evolutionary adaptations to a range of ambient temperatures in amniotes.