The MRCPPU CRISPR facility has a wealth of experience with >900 successful projects completed over the past 5 years comprising KOs and KIs in a variety of cell types including standard adherent cancer lines, mouse ES cells, and human iPSCs.
Our bespoke service provides:
- Core design
- Detailed maps
- Cloning of required guides and donor plasmids
- Genotyping primers
- Detailed protocols
- Technical advice
All cell-line generation steps are performed by the end-user using the detailed protocols provided.
We opt for a paired nickase plasmid-based system, where possible, to minimise off-targeting and for each project 2 guide pairs are provided to maximise the odds of successful targeting 1, 2. Each guide pair is designed to have a low combined off-targeting score and care is taken to ensure no off-target sites exist within 1 Kb to one another such that non-specific cleavage is considered negligible and resulting lines have a more reliable phenotype. Guides may be cloned into alternative systems expressing wild-type spCas9 where necessary e.g. lentiviral expression plasmids to allow for more difficult targeting such as delivery into primary lines.
- Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389, September 12
- Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc. Nov;8(11):2281-308.)
If you are interested in using the service please contact Tom at tmacartney@dundee.ac.uk to arrange a chat to discuss the proposed project.