CRISPR

CRISPR @CEDOC: Affordable CRISPR/Cas editing of cell lines

We are a research group working at the Chronic Diseases Research Centre of Nova Medical School, Portugal. The group leader has been publishing for several years on the RAG1/RAG2 endonuclease- and Activation-induced deaminase-dependent processes underlying the immunoglobulin gene rearrangements that result in the production of improved antibodies by B cells (e.g. 1-5). As these processes involve the generation of double-strand breaks in the antibody genes, the CRISPR-Cas technology was appealing to us. Like hundreds of groups around the world, we adopted CRISPR-Cas as our preferred gene editing technology to manipulate the genome. In parallel, we also became interested in the technique itself, and we’ve been working on ways to improve it (6). Given the prohibitive prices charged for genome editing services by companies, we decided to create a service with competitive prices and appealing options. The service was officially launched in October 2019 and we have started dozens of projects.

References
1. Nature Communications (2014) 5623
2. Front Immunol.(2013) 16 4:110
3. G3 (2013) 3: 645-655
4. J. Exp Med (2005) 6:733-8
5. Mol Cell 12: 501-508
6. BioRxiv (2019)

 
Countries that have purchased our services:

 
How to order your cells
We’ll schedule a video conference to discuss your project at length, understand your goals and decide what type of service suits you. Once we have an agreement and we receive the upfront payment (25% or 50%, depending on the package), the clock starts ticking (the typical turnaround time is 5 months). We begin immediately designing the approach and producing the required constructs. At the same time, we will arrange the transfer of the cells from your lab to our facility (Note: if your budget is small, you may choose to ship the cells at room temperature using this protocol. You pay the remaining 75% when you receive the modified cells.

For further details, write to polina.kharitonova(at)nms.unl.pt.

 
Terms of agreement
1. You pay 25% of the service upfront. You pay the remaining 75% when the clones or bulk cultures are ready to be shipped. In the case of the “do it yourself” option, you pay 50% upfront and the remaining 50% when the project is concluded or when 4 months have passed since the date the cells were shipped to you (even if the project was unsuccessful).

2. If we fail to deliver the clones or cultures within 4-7 months, we are obliged to show you evidence that the planned tasks were performed and that different attempts were tried to surmount the technical problems.

. In case of failure to transfect the cells, we’ll show evidence of attempts using electroporation (Amaxa and/or Neon), calcium phosphate transfection and cationic lipid-mediated transfection (e.g. Lipofectamine).

. In case of failure to introduce Cas9-dependent breaks in the locus, we’ll show evidence of trials using the included set of gRNAs and an extra set of gRNAs.

. In case of failure to insert an SNP or sequence, we’ll show genotyping evidence and trials using a different template DNA (e.g. oligos and single-stranded rAAV).

. In case of failure to isolate single clones, we’ll report the problems during the limiting dilution step.

3. If we did not fully explore all the included tasks and failed to deliver the clones or cultures within 4-7 months but we believe it will be possible to solve the problem in two extra months using the included tasks, if you agree you can still have the edited cells and you pay only 75% of the service in total (i.e., you’ll pay 50% upon delivery of the cells, instead of the remaining 75%). If you do not agree, you’ll be reimbursed of the 25% you paid upfront.

4. If we exhausted the included tasks and failed to deliver the clones or cultures within 7 months but we believe the problem is solvable using technology not included in the original package (e.g., the use of a Cas9 fused to a monomeric streptavidin and a biotinylated template oligo, or a transduction protocol), we’ll readjust the budget and make a new proposal. If you do not agree, you will not be reimbursed.

5. You’ll receive a paragraph that will include a reference to our service and the relevant papers. You’ll include this paragraph in the materials and methods of your manuscript(s) and you may change the style and content, but always keeping the references to the service and the papers.