CRISPR Service

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.

  1. 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
  2. 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.

 

 

Do you need any help? Please get in touch and we’ll be happy to lend a hand.