Gene Editing (CRISPR-Cas9)
The Stem Cell Core Facility has successfully generated CRISPR-engineered lines with various modifications:
- Knock-in (generation of reporter lines by adding a fluorescent tag to your gene of interest; introduction or correction of point mutations to make isogenic lines), as seen in
- Garcia-Diaz A., Efe G., Kabra K, Patel A, Lowry E.R., Shneider N., Corneo B.,Wichterle H. Standardized reporter systems for purification and imaging of human pluripotent stem cell-derived motor neurons and other cholinergic cells. Neuroscience, 2020 Jun 30:S0306-4522(20)30404-8.
- Knockout (with donor DNA if deletion/insertion of specific sequence of nucleotides is required, or without donor DNA, in which case loss of function will result from the random insertion/deletion of base pairs causing frame-shift mutations)
We use RNPs, with synthetic gRNAs and Cas9 protein.
- Gene name
- Gene ID
- Desired mutations
- Cell line to modify (only lines that have been confirmed as mycoplasma-negative by the customer)
Pricing varies depending on the level of involvement of the Stem Cell Core Facility staff. We can either take over the targeting project from start to finish, or otherwise be involved in only certain steps, as some customers may prefer to perform some steps by themselves (e.g., genotyping).
All projects performed in their totality by the Stem Cell Core Facility staff include:
- Design of guide RNA and donor constructs (no donors required for knock-out via NHEJ)
- Targeting of the desired cell line
- Genotyping of clones to assess hetero- or homozygosity of recombination
- Cloning of the targeted alleles into vectors for proper sequencing
- Expansion of positive clones
Time for completion: 3-4 months (estimated)
- Our facility focuses only on targeting of hESC & hiPSC.
- If guides are supplied by the customer, we strongly advise you to test them prior to the targeting of the desired cell line (this can be done by the customer or by the facility for an additional charge)
- The only goal of the facility is to provide evidence of proper gene targeting. We do not perform functional assays of the positive clones unless an optimized protocol that is established and routinely performed at the facility is applicable (at an additional price).
- Waiting period until the start of a project might be applicable.