This laboratory-based training course will provide a comprehensive overview of the theory and practical laboratory skills required for the genetic manipulation of mammalian stem cells, focusing on human iPS cells. Genome informatics, vector construction and genome editing strategies using CRISPR-Cas9 will be covered in interactive practicals and demonstrations, with discussions and lectures by international experts.
Laboratory work includes the use of CRISPR-Cas9 systems for advanced genome engineering and genetic screens, the design and construction of gene targeting vectors, the culture and transfection of human iPS cells and the derivation of iPS cells and organoids.
An emphasis will be placed on the integrated planning, design and successful experimental execution of gene targeting projects, allowing participants to gain the skills necessary to design, construct and target their own genes of interest.
The programme will include lectures and practical laboratory/computer-based sessions covering the following topics:
1. Informatics
The visualization and interpretation of gene models will be presented focusing on the practical design of gene targeting strategies, including CRISPR gRNA selection and the design of oligonucleotide and other HDR substrates. Students will also have the opportunity to develop their own CRISPR-mediated gene targeting designs using web-based tools and learn how to exploit public genomic data resources.
2. CRISPR Gene Targeting Vector Construction
Students will learn to apply recombineering and synthetic biology techniques to gene targeting vector construction and engage in the design, assembly and analysis of vectors for CRISPR-assisted gene targeting applications; including protein tagging approaches.
3. Stem Cell Culture/Genome Editing
Genome editing experiments will be performed where participants will learn feeder-free culture of human iPS cells and transfection techniques for protein-based delivery of Cas9 to engineer a variety of useful alleles such as knockouts, introduction of SNPs and tagged lines. Cell manipulation techniques including picking, expansion, archiving and genotyping of iPS cell clones will also be covered.
4. Genome-wide screens
Practical application of the CRISPR system to undertake in vitro genetic screens will also be explored. This includes the design of genome-wide screens, lentiviral transduction of mouse ES cells and bioinformatic analysis of next-generation sequencing data.
5. iPS Cell Generation
Participants will be introduced to protocols for the derivation of high quality iPSCs, using non-integrative reprogramming techniques, required for the characterisation, differentiation and genome editing of human iPSCs.
6. Organoid derivation and differentiation
Advances in 3D cell culture techniques have facilitated the derivation of organoids that model a range of tissues, including brain and lung. Experimental approaches for the derivation of these organoids will be discussed, together with their potential applications for studying cellular interactions, tissue organisation and disease progression.
Learning Outcomes
After attending this course, participants should be able to:
