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

A.Y. 2019/2020
3
Max ECTS
42
Overall hours
SSD
AGR/03 AGR/07 AGR/12 BIO/01
Language
Italian
Learning objectives
This course is aimed at providing the students with knowledge on genome editing and its potentiality in crop improvement.
Expected learning outcomes
This course will provide the students with knowledge in the following areas:
1. identification of target genes useful in crops protection
2. planning of genome editing experiments
3. analysis of genomic sequences derived from genome editing events
4. in vitro culture setting and lab experience
5. crop pathology phenotypization
Course syllabus and organization

Unique edition

Responsible
Lesson period
Second semester
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 work
Bibliography
A lecture note is provided during the course
Assessement methods and criteria
Students develop a project independently and present it to the course teachers
AGR/03 - ARBORICULTURE AND FRUITCULTURE - University credits: 0
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