97028 - Genome Engineering for Functional Genomics

Learning outcomes

At the end of the course, students will become familiar with the advances in genome engineering technologies that enable the precise control over genome sequence and regulation. They will know multiple approaches to functionally characterize and manipulate genomic information. Students will learn the practical aspects of the technologies used to modify human and model organisms’ genomes, by choosing the appropriate molecular tools to investigate the function of single or multiple genes modified at once.

Course contents

1) From Gene Targeting to Gene Editing: the steps to the modern concept of genetic engineering.

2) Considerations on engineering model systems to model human disease.

3) The basic elements of the toolbox (LoxP, Frt, Rox, Att sites and respective recombinases) and how to assemble them in DNA vectors (Gateway technology, Gibson assembly, Golden Gate technology, Recombineering). Modular assembly of targeting vectors.

4) Retrieving gene and genome information (Genomes databases) and software design tools (e.g. Gene Construction Kit, SnapGene).

5) Genetic modifications and strategies to obtain genetic Knock-Out (KO):

how to achieve a gene KO (deletion of a critical exon, early insertion of a reporter, complete deletion of coding portion of a gene; achieving gene KO by Gene Editing (with one, two or multiple guide directed nucleases).

6) Genetic modifications to introduce specific mutations: in vitro mutagenesis, in vivo targeting vectors and donor vectors.

7) Conditional expression in vitro and in vivo: TAT-cre and TAT-dre recombinant proteins, tamoxifen inducible Cre, tetracycline inducible expression systems (Tet/tTa), light controlled reporters.

8) Studying in vivo the expression of a gene. Different types of reporters. Linking the expression of a gene with a reporter: ires elements and 2A self-cleaving peptides. Lineage-fate studies of stem cell marker genes.

9) Studying the effect of chromosome rearrangement and of chimeric/fusion proteins: Knock-In transgenic models (safe harbours loci, e.g. mouse Rosa26 locus), mimicking chromosomal translocation and induction of chromosomal translocation in vivo.

10) Introducing multiple genetic changes at once: multiplex genome engineering.

11) Somatic genome engineering: targeting specific tissues and organs in vivo or ex vivo.

12) Whole genome engineering by synthesis: the Genome Write project.

Readings/Bibliography

The material of the lectures and scientific articles from the literature will be provided to students.

Teaching methods

The course is structured in lectures (16 hours) in the classroom

Assessment methods

Student learning will be checked at the end of the course with an oral exam. During the course, students will be asked to give a presentation discussing a scientific article taken from the literature. The presentation will be evaluated.

Due to the COVID-19 restrictions, exams could be held at distance on Microsoft Teams platform.

The final mark will be calculated as the weighted mean (based on CFUs) of the marks obtained in all the courses of FRONTIERS IN MEDICAL RESEARCH (I.C.).

A mark above or equal to 18/30 in each module of the Integrated Course is required to obtain the final assessment.

Teaching tools

Additional material will be provided to supplement lectures. Scientific articles and additional material will be provided to students for further reading.

Office hours

See the website of Emanuele Panza