Adapting CRISPR-Cas9-derived tools for site-specific genome engineering
Various CRISPR-Cas9-derived tools enable genome engineering, allowing efficient gene knock-out, knock-in, and site-specific epigenetic tuning of expression. The course will provide hands-on training in genome editing using a couple of generations of CRISPR-Cas9 tools. Thus, to begin with we will train to bioinformatically design the necessary tools, to produce the sgRNA of interest using plasmid expression vectors and to use ribo-nuclear protein (RNP) complexes (sgRNA-Cas9 complexes as mode of igniting genome editing). We will knock-out, and in a second experiment enable one base-pair edit at a specific genomic locus. In his course transfection will be used side by side with electroporation to appreciate the importance of their optimization for efficient engineering outcomes. Assays for detecting site-specific genomic modifications will be employed.
To accompany the practical part, we will review the emergence of CRISPR-derived technologies as a vital discipline relevant for research and biotechnology in cells and organisms alike. In addition, we will learn to appreciate the current, yet limited, knowledge of the cellular DNA repair machineries that underlie efficient genomic modifications and the basic knowledge required to allow optimal delivery, specificity and fidelity that a vital for successful editing.
Finally, participants are expected to design a project of their own, considering in-silico parameters as well as experimental ones, present it and conduct an experimental notebook throughout the course duration that will be presented at the end of the course. If relevant, selected participants will have the option to generate tools of choice (e.g. plasmids) to allow them to initiate their own projects back at their mother laboratories.