Ree Crispres - Using Crispr-Based Genomic Editing for Crop Protection

Time required: 7 days, 50 hours of laboratory work; 22 hours for the preparation of the final report; 3 hours for the evaluation of the final report (total: 3 CFU).
DAY 1 (8 hours). Toffolatti
· Introduction on plant-pathogen interaction (2 hr)
· Plasma-experimental experimental inoculations (3 hrs)
· Microscopy (3 hrs)
o Stereo microscopy on plasma screen
o Fluorescence microscopy (NoLimits platform).
DAY 2 (6 hours). WELLS
· Introduction to new breeding techniques and contextualization of genome editing.
· Theoretical introduction on susceptibility and knock-out genes
DAY 3 (7 hours). DE LORENZIS
· Introduction to in vitro cultures.
· Cultivation of phytomers by in vitro micropropagation.
· Cultivation of axillary buds for the production of somatic embryos.
· Identification and selection of somatic embryos in the globular phase.
DAY 4 (8 hours). BRAMBILLA
· In silico identification (computer lab) of the best gene targets for a genome editing experiment focused on the protection of plants of agricultural interest (4-5 hrs)
· Selection of guide RNAs (use of software such as Benchling);
· preparation of oligonucleotides;
· assembly of the virtual construct.
DAY 5 (8 hours). BRAMBILLA
· transformation of E. coli with prepared constructs;
· transformation of A. tumefaciens;
· agro-infiltration of leaves;
· screening of transformants by PCR.
DAY 6 (7 hours). DE LORENZIS
· Evaluation of potential off-target (NGS and Sanger)
· introduction to the use of HRM PCR for the identification of off-targets;
· sequence analysis
DAY 7 (6 hours). ALL
· Identification of projects for students
· Indications about the presentation / final report
· Subsequently, examination by the teachers of the projects prepared by the students and selection of the project to be presented as an example of the activities of the CRISPres laboratory