|teoria||4||I semestre||Barbara Molesini|
|laboratorio||2||I semestre||Barbara Molesini|
At the end of the course, the student will acquire the theoretical and practical basis for the design of various types of genetic constructs aimed at developing transgenic, cisgenic, and intragenic plants. In addition, genome editing tools will be covered. Attention will be paid to the application of these strategies for qualitative and quantitative crop improvement.
Molecular mechanism of stable transformation via Agrobacterium: current knowledge of the process of T-DNA integration into the plant genome; transgene integration, stability, methylation and silencing; strategies to avoid transgene silencing; promoters used for genetic construct preparation (constitutive, spatiotemporal, inducible, synthetic); cis–trans engineering; synthetic circuits; multigene engineering; artificial miRNA and target mimicry; cisgenesis and intragenesis; cisgenic and intragenic genetic constructs; strategies to remove marker genes from transgenic plants; artificial programmable DNA nucleases (ZFNs and TALENs) and RNA-guided DNA nucleases (Type II CRISPR-Cas9 of Streptococcus pyogenes) for genome engineering; CRISPR-Cas9 mechanism of action; sgRNA design; minimization of off-target activity; dead Cas9: application of the CRISPR-Cas system beyond genome editing; genetic constructs for genome engineering using the CRISPR-Cas9 system, screening of mutants generated by CRISPR system.
Supplied educational material:
Power point lessons, relevant research articles and reviews.
1) In planta application of RNAs coupled to nanoparticles for the silencing of target transcripts;
2) Design of guide RNA using free bioinformatic software, in vitro transcription of guide RNAs for targeted genome editing and screening of the most effective guide RNAs for the cleavage in vitro of their targets in the presence of Cas9.
The exam will ascertain the students’ knowledge on the topics of the lectures and lab practices. The exam, for both attending and non-attending students, will consist in a written individual exam featuring open-ended questions and exercises.