Ree Crispres - Using CRISPR-based genomic editing for crop protection

A.Y. 2020/2021
Overall hours
AGR/03 AGR/07 AGR/12 BIO/01
Learning objectives
L'obiettivo del corso è quello di fornire conoscenze sul funzionamento del Genome editing e le sue potenzialità nelle specie d'interesse agrario.
Expected learning outcomes
L'attività integrativa prevede di fornire competenze agli studenti nei seguenti ambiti:
1) Identificazione dei geni target per la protezione delle piante di interesse agrario
2) Pianificazione di esperimenti che utilizzino genome editing
3) Analisi di sequenze derivanti da eventi di editing genomico
4) Colture in vitro
5) Fenotipizzazione della malattia
Course syllabus and organization

Single session

Lesson period
Second semester
The REE laboratory will be conducted in shifts that allow the simultaneous presence of a number of students compatible with current regulations. Compatibly with the health situation, the project developed by the students and selected by the teachers will be presented during a dedicated event.
Course syllabus
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
Prerequisites for admission
Basics in genetics, molecular biology, plant pathology
Teaching methods
Lectures and lab works
Teaching Resources
A lecture note is provided during the course
Assessment methods and Criteria
Students develop a project independently and present it to the course teachers
AGR/07 - AGRICULTURAL GENETICS - University credits: 0
AGR/12 - PLANT PATHOLOGY - University credits: 0
BIO/01 - GENERAL BOTANY - University credits: 0
Laboratories: 36 hours
Lessons: 6 hours