This laboratory-based workshop will provide a comprehensive overview and laboratory experience of advanced genome engineering of human and yeast cells using CRISPR/CAS. This week-long workshop will consist of a series of integrated lectures, interactive demonstrations, discussions .and experiments in which participants learn how to design CRISPR/CAS DNA vectors for genome manipulation. CRISPR/CAS will be compared with alternative genome engineering methods including TALENs and zinc finger nucleases. An emphasis will be placed on the planning, design and successful experimental execution of gene targeting projects. During the workshop participants will engineer genomes of mammalian cell and yeast using CRISPR/CAS9. The latest innovations in CRISPR technology will be explored.
- How to design gRNAs and knock-ins using available online design tools.
- Practical rules to increase efficiency of knock-outs and knock-ins, including accounting for species-specific differences.
- Generation of guide RNA (gRNA) using in vitro transcription and expression plasmids.
- Assays for detecting successful genome manipulation and validation of clones.
- Basics of targeted mutagenesis.
- Understanding specificity and off target effects, including latest improvements to improve specificity.
- Homologous recombination and the role of host recombinases.
- Genome-scale transcriptional control: use of transcriptional activators and represssors.
- Editing of stem cells: iPSCs, ESCs, MSCs and HSCs.
By the end of this workshop, participants should gain the skills necessary to design, construct and target their own genes of interest in human cells and yeast cells.
Target audience: individuals with biology research backgrounds and similar professionals