My project involves optimization of CRISPR/Cas9 methodology of genome editing in plants. CRISPR/Cas9 is a method of choice to perform genome engineering. There are however significant limitations which prevent broader implementation of this technology in plants.
These limitations include variable efficiency of editing at different targets, off target activity, inefficient inheritance of the created mutations, ability to edit simultaneously several targets, limited selection of targets/PAM repertoire and the need to segregate Cas9 and sgRNA from the created mutations. Numerous configurations of CRISPR/Cas9 designed to address these limitations had been published. Our aim is to establish a uniform testbed and toolkit, where many of these configurations are tested under the same conditions and their editing efficiency and off target activity will be assessed. In order to minimize variability in transgenic expression we established editing essay in plant protoplasts.
Our experimental design includes transforming protoplasts from the same harvest with different configurations of CRISPR/Cas9, including Cas9 variants which specifically edit NGG, NGAG, NGCG and NNGGGT PAMs , Cpf1s which recognise TTTN PAM, and SpCas9 variants with reduced off target activity, and assessing frequency of indels and double stranded breaks activity employing DNA capture assays and Next Generation Sequencing. Currently we gained experience in efficient extraction and transformation of the protoplasts from different plant species using our CRISPR/Cas9 constructs and we are establishing high throughput protoplast transformation methodology using automatic dispenser. In the next step we will attempt to regenerate plants from the edited protoplasts. We also trying to find the ways to perform successful CRISPR/Cas9 assisted targeted repair of gene of interest. We follow the two-step strategy: transforming the plants with “landing pad” with subsequent insertion of the repair template. Successful insertion of the repair template should restore the herbicide resistance and facilitate selection of the plants with successful repair.
I participate in the proposal for Open Plant funding titled “Establishing Low Cost Microfluidic System for Single Cell Analysis” (Dr. Steven Burgess is a principal applicant). The aim of the project is to establish cost-effective microfluidic device for single cell sorting and analysis. Significant reduction of the cost comparatively to the commercially available systems is achieved by producing some of the parts of the device such as microscope and syringe part with 3D printing technology and utilizing open source materials and repositories. Among various applications for this device will be sorting the transformed protoplasts according to the cell size and strength of the fluorescence of the transgene, and cost-effective miniaturizing and automatizing Golden Gate cloning assembly reactions.