We utilise CRISPR/Cas9 technology to manipulate the host’s genome, in order to understand the function of a gene of interest (GOI). In a simplest installation, a choice of selected single guide RNAs (sgRNAs) will direct the Cas9 nuclease to a sequence-specific site on a GOI, introducing indels through nonhomologous end joining (NHEJ), leading to a complete gene inactivation. If knock-out is viable, we can assess its phenotype in comparison to its wildtype counterpart. This in turn will yield information on the changes that occur upon loss of function of a GOI validating target’s involvement in a studied cellular pathway or a disease model. Knock-out can also be used to confirm engagement of a small molecule with a target, as well as its off-target effects.
Additionally, a combination of an sgRNA and a DNA template, fuelled by homology-directed repair (HDR), can allow us to precisely edit a defined endogenous amino acid(s) with an assumed function, augmenting final gene product, system’s phenotype, and/or specificity of a given small molecule binding to a target.
We can introduce indels, point mutations, tags, and protein markers via CRISPR/Cas9-mediated NHEJ or HDR to validate targets and small molecules engagement in cells, complemented by a choice of downstream readout assays (including gene-reporters, immuno-assays, imaging analysis).
o2h team can:
- Design sgRNAs and DNA repair templates
- Perform gene editing in mammalian cells and generate clonal population
- Assess the phenotype of knock-out or knock-in cells with a choice of redouts (including gene-reporters, immuno-assays, imaging analysis)
- Validate targets and their engagement with small molecules
- Conduct functional mutagenesis analysis of the endogenous amino acids
- Introduce tags and markers on an endogenous protein for downstream application in biochemical, biophysical or imaging analysis
To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.
