Project description:Plant biotechnology needs new methods that accelerate design-build-test-learn cycles to develop new gene editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle –from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors and used it to develop an optimized LbCas12a-ABE system. Sequential improvement of five components –NLS, crRNA, LbCas12a nuclease, adenine deaminase and linker– led to a systematic, stepwise increase in ABE activity at extrachromosomal GFP reporter (from 0.5% to 40%) and endogenous target sites. We confirmed the activity of LbCas12a-ABE in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.
Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.
Project description:ra10-02_viromouv - viromouv - transcriptome analysis of Arabidopsis Companion cells after infection by plants viruses - Transgenic plants expressing GFP under the control of a companion cell specific promoter were infected by two different viruses: LMV and TuYV. Companion cell protoplasts from these plants were sorted by FACS and extracted RNA were compared to healthy companion cell protoplasts.
Project description:The goal of this experiment was to investigate the early transcript changes (6h) induced by hypoxia treatment in mesophyll protoplasts. A single pair (control & hypoxia) of GeneChips® was used to confirm that hypoxia treatment altered the expression of an overlapping set of genes controlled by KIN10 (At3g01090) in Arabidopsis mesophyll protoplasts. Keywords: KIN10, KIN11, darkness, hypoxia, starvation, stress, sugar signalling, Arabidopsis, SnRK1
Project description:Firstly, wild type seedlings that had germinated in root tubes for 14 days were taken to prepare protoplasts. The GCN5-FLAG transient expression vector was transformed into wild type protoplasts. The protoplasts were collected and purified by Flag beads. After quality testing of the samples, phosphorylation mass spectrometry sequencing of GCN5 was performed.
Project description:To investigate whether the oxidative stress response is induced in protoplasts, we compared the gene expression patterns of protoplasts to those of walled cells and L-forms using microarrays.
Project description:ra03-03_protoplats - transcriptome profiling from a protoplast culture of arabidopsis thaliana - transcriptome profiling and comparison of 6 status of differentiation - protoplasts were extracted from plantlet cultivated during 2 weeks then placed in a culture midium which stimulate cell division. Protoplasts were harvested after 0, 24, 48, 96 or 168 hours of culture. Biological repeat has been done (experiments A and C) Keywords: time course