96032 - Crispr-Cas Systems: from Bacterial Adaptive Immunity to Genome-Editing Applications

Learning outcomes

The student will gain in depth knowledge of the molecular mechanisms governing adaptation, expression and interference of the major CRISPR/Cas bacterial immunity systems; of the CRISPR/Cas-based methodologies used for genome editing and gene-expression modulation; of the main CRISPR/CAS-based biotechnological applications. In particular, at the end of the course, the student will be able to: - analyse and discuss the main topics concerning CRISPR/Cas mechanisms and applications; - understand, analyse and discuss research papers; - plan experimental approaches to study a biological problem.

Course contents

Biology and mechanisms of CRISPR/Cas-based adaptive immunity in bacteria.

From the first observations to the characterization of the CRISPR-Cas system as an adaptive bacterial immune system.

General features and key points of the system. Classification and complexity of the various CRISPR systems observed to date.

The different stages of CRISPR-Cas immunity: acquisition of new spacers (immunization), expression of the locus and processing of the precursor transcript(s); interference.

Detailed description of the interference mechanism of the most studied systems (Cas9; Cas12; Cas13; Cascade-Cas3).

CRISPR-Cas system applications.

Applications of CRISPR-Cas systems for the generation of double-strand breaks on DNA: gene-editing techniques based on the Non-Homologous End Joining (NHEJ) mechanism; gene-editing methodologies based on Microhomology-Mediated End Joining (MMEJ) and Homology-Directed Repair (HDR) mechanisms. Comparison with other tools currently used for genome editing, including Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

Genome-editing applications that do not involve the generation of double-strand breaks on DNA: base editors, prime editors.

Use of CRISPR-Cas systems for the modulation of gene expression in prokaryotic and eukaryotic organisms (CRISPRi / CRISPRa); epigenetic engineering technology (CRISPRoff).

Beyond genome editing: CRISPR/Cas-based biotechnological applications.

Ethical issues.

Readings/Bibliography

Reviews and seminal papers.

Teaching methods

Lessons, analysis and in class discussion of seminal research papers.

Summarizing PowerPoint presentations.

Problem posing and solving sessions, student workgroups, flipped classroom approaches, active learning exercises.

Assessment methods

The final exam will verify the acquisition of the main learning outcomes. In particular, the student will be able to:

1) analyse and discuss in detail the main topics about CRISPR-Cas mechanisms and their applications;

2) understand, analyse and discuss research papers;

3) plan experimental approaches to study a biological problem.

The final exam is made of two parts. Part one: analysis and presentation of a seminal research paper (learning outcome 2, max 10 points); part two: oral exam (assessment of learning outcomes 1 and 3, max 20 points). The various partial marks of the oral interview, combined with the evaluation of the oral exposition of the scientific article, contribute to the final grade.

Teaching tools

Reviews and seminal research papers. PowerPoint presentations. Registered students can download lesson presentations, articles and other teaching materials through the Insegnamenti Online website: https://virtuale.unibo.it

Office hours

See the website of Davide Roncarati