SVEK23001U CRISPR Tsunami: Design and Hands on Gene Editing
MSc Programme in Biotechnology - restricted elective
MSc Programme in Molecular Biomedicine - restricted elective
MSc in Human Biology - elective
The bacterial immune defense system CRISPR has proven to be extremely efficient for gene and genome editing. Genome editing is critical in a huge number of applications in biology, ranging from medicine, therapeutics, and biotechnology. The CRISPR gene editing field is a prestigious and extremely hot research area evolving at a tremendous speed and with constant implementation of technological advances. The Nobel prize awarded to the field in 2020 boosted this “tsunami” even further.
Common for all applications of CRISPR for gene editing is the need for computational-based design of guide RNAs (gRNAs) to select the ones which are expected to work most efficiently. The designed gRNAs are subsequently delivered to the target cells or organisms and be expressed together with the Cas protein machinery performing the manipulations. Following the gene editing the specificity of the manipulations must be assessed.
This interdisciplinary course will introduce hands-on application and theory of the computational tools for gRNA design followed by experimental techniques applying these gRNAs in manipulations of immortalized mammalian cells, bacteria as well as in whole organisms (zebrafish). On the computational side cutting-edge tools for on- and off-target gRNA design for Cas9 (the most widely used CRISPR enzyme) will be covered at a level matching course participants that have a molecular biology background. On the experimental side gene editing and silencing will be performed in the different organismal contexts including (1) the human pathogen, Staphylococcus aureus involving design of constructs, transformation, and assessments of expression of targeted genes, (2) mammalian cells where the course participants will introduce mutations leading to Alzheimer’s disease to create an in vitro disease model and (3) whole vertebrate organisms (zebrafish) where the course participants will introduce knockouts, which lead to albinism.
Post gene editing it is critical to assess the specificity of the manipulations, that is, if the intended on-target was obtained without any off-target effects. State-of-the-art computational methods for such post editing analysis will be introduced.
The students will have deep understanding of
- the CRISPR system and how it is used for gene editing
- the design process of editing experiments
- post analysis for quality control after the editing has been carried out
The students will be able to do
- hands-on design of CRISPR experiments using computational tools
- genetically manipulate human bacterial pathogens, mammalian cell lines and whole vertebrate organisms (zebrafish)
- Understanding of the computational methods behind gRNA design
- Understanding of the experimental techniques involved in CRISPR based genetic manipulations
- Critical assessment pros and cons of computational CRISPR design and analysis tools
- Hands on experience and practical knowledge in performing CRISPR gene editing in mammalian cells, zebrafish and bacteria
The literature is listed in absalon and will be based on research articles.
Basic knowledge of biochemistry.
Basic knowledge of genetics and cell biology.
Previous practical experience in pipetting and handling cells or bacteria.
- Class Instruction
- Theory exercises
- Practical exercises
- 7,5 ECTS
- Type of assessment
- Written assignment, 2 weeks
- Type of assessment details
- A report on your analyses should be worked out containing the
following sections and content:
* Title of the report
* Name and KU identifier
* A max 300 character abstract
* Introduction: Justification of the problem and choice of methods for the analyses carried out.
o Conceptual description of the methods used.
o Justification for the how methods are used.
o The outcome of gRNA design
o Experimental analysis of successful gene editing via restriction polymorphism analysis
o Highlight the main outcome
o Describe which parts of the analyses that might have some uncertainty and how that may have influenced the outcome of the work and which parts of the results may be the most uncertain ones.
o Provide a conclusion (the last paragraph)
Report format requirements:
A report template will be provided and must be used for the report:
The report must not exceed 20000 characters (including spaces and references)
A maximum of 4 figures and/or tables, which must be placed after the 20000 characters starting with a new page.
References must be placed in the end of the report
The report must be handed in by the end of the last week of the course.
- All aids allowed
- Marking scale
- 7-point grading scale
- Censorship form
- No external censorship
- Exam period
The last two weeks of the block
A new written report must be handed in.
Criteria for exam assesment
The report will be assessed relative to Learning Outcome and the students’ possible accompanying innovative approach.
- Course code
- 7,5 ECTS
- Full Degree MasterFull Degree Master choice,Ph.D.
- 1 block
- Block 3
- Course capacity
- Study Board for the Biological Area
- Department of Veterinary and Animal Sciences
- Faculty of Health and Medical Sciences
- Jan Gorodkin (8-6a727572676e6c714376787167316e7831676e)
Louise von Gersdorff Jørgensen