Various CRISPR-Cas9-derived tools enable genome engineering, allowing efficient gene knock-out, knock-in, and site-specific regulation of epigenetic events. The course will provide hands-on training in genome editing using 3 generations of CRISPR-Cas9 tools. Thus, Cas9-expressing plasmids, Cas9 RNA, and the Cas9 protein will be employed, along with an appropriate single-guide RNA (sgRNA) to knock-out, knock-in, and edit at specific genomic loci. This course will emphasize training to design the necessary tools and to produce the sgRNA of interest and Cas9 RNA by in-vitro transcription as well as using ribo-nuclear protein (RNP) complexes. Transfection will be used side by side with electroporation to appreciate the importance of their optimization for efficient engineering outcomes. Assays for detecting gene modifications and clone validation will be employed.
To accompany the practical part, we will discuss various relevant topics, and to name just a few, we will study the emergence of several CRISPR-derived technologies as a vital discipline relevant for research and biotechnology in cells and organisms alike. This includes the current, yet limited, knowledge of the cellular DNA repair machineries that underlie efficient genomic modifications and the basic knowledge required for efficient gene knock-out and knock-in that should be characterized by minimal undesired “off-targets”.
Finally, participants will design their sgRNA of choice, produce it, and they will generate sufficient Cas9 RNA to allow them to initiate their own projects back at their mother laboratories.