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.

Limited consequences for loss of RNA-directed DNA methylation in Setaria viridis domains rearranged methyltransferase (DRM) mutants

The Domains Rearranged Methyltransferases (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. There is also evidence for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions do not have altered expression in the drm1ab mutant even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.

Plant remains unearthed at the Donghulin site in Beijing: discussion on results of flotation

A number of charred plant seeds were recovered from the Donghulin site by means of flotation. The site is located in suburban Beijing and dates from 11,000 to 9000 BP. A total of 14 charred grains of foxtail millet have been collected and identified as of the domesticated species (Setaria italica) according to morphological analysis. One grain of broomcorn millet was also identified. These are the earliest domesticated millet grains recovered by flotation, providing crucial archaeological evidence for understanding the timing, locations, and processes of millet domestication. Moreover, the charred seeds of Setaria viridis provide important clues for exploring the wild ancestral plants of foxtail millet and the domestication process. The results of flotation at the Donghulin site are important for understanding the origins of dryland agriculture in North China, which was predominated by millet farming.

Physiological responses of Agriophyllum squarrosum and Setaria viridis to drought and re-watering

Drought resistance of psammophyte determines survival and growth, but their responses to drought are not well understood. We conducted a pot experiment to study how physiological characteristics respond to drought and rehydration. We found that watering to 60–65% of field capacity (the control) provided more water than was required by Agriophyllum squarrosum and its leaves became yellow and slightly wilted. The total chlorophyll content and Fm (maximum fluorescence after dark adaptation) in control were lower than in the drought treatment, and both decreased after rehydration. With increasing drought duration and intensity, the relative water content (RWC), chlorophyll content, Fm, and the quantum efficiency of photosystem II (Fv/Fm) of Setaria viridis decreased, but malondialdehyde and membrane permeability increased. During the late drought, the activities of three antioxidant enzymes in A. squarrosum increased to prevent membrane lipid peroxidation; for S. viridis, only peroxidase and superoxide dismutase activities increased. After rehydration, RWC of both species increased, but Fv/Fm of A. squarrosum and Fm of S. viridis did not recover under severe drought. Our research illustrated that A. squarrosum is better adapted to arid environment than S. viridis, but the high soil moisture content is not conducive to normal growth of A. squarrosum.