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. We now offer our expertise to researchers within the university of Dundee.

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.)
  • £350
  • 2 weeks : Design, cloning and delivery of components.
  • 4-5 weeks: CRISPR process from transfection to cell-line verification.

We recommend screening using specific antibodies where possible as this allows the CRISPR success rate to be determined within 5 days of transfection following an immunoblot of the cell pool(s). If the transfection rate is good and the gene is non-essential* then it is relatively easy to obtain the desired KO cell-line using our protocols, researchers should expect 2-3 homozygotes from a screen of 10 clones. The success rate drops to 1 in 10 if 3 copies of the target chromosome are present (most cancer lines tend to fall into this category), and drops further again with each additional copy. *The KO/knockdown of essential genes may be accomplished by the KI and fusion of elements such as GFP or BRD4 to allow degradation using AdPROM or Protacs in a time-dependent manner.
  • £850 for inserts less than 1 Kb (£300 per additional Kb)
  • 4-6 weeks: Design, cloning and delivery of bespoke donor and guides.
  • 4-5 weeks: CRISPR process from transfection to cell-line verification.

HDR is rare thus a fluorescent reporter is required to allow enrichment by FACs, donors generally consist of a bicistronic cassette separated by an IRES2 element. KI efficiency varies depending on the target site, cell-line and size of the donor though researchers should expect 1-2 homozygotes from a screen of 20 clones following sorting for GFP. For hypotriploid cancer lines with >2 copies of the target chromosome the most realistic outcome is a KI into 1 or 2 alleles with any additional chromosomes being KOd by indel formation.
Point mutations in internal exons are possible though extremely difficult to achieve since a non-fused reporter cannot be used. Options are available, though success is not guaranteed.

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.

 

 

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