CRISPR Tsunami: Design and Hands on Gene Editing

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.