CRISPR-Cas9 is a technique used for genome editing that is adapted from the bacteria’s antiviral immune response. Bacteria capture and store DNA fragments from invading viruses within a region of their genome, and these CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) guide sequences help detect and protect the bacteria from future infections. When the CRISPR guide sequences detect an invading virus or DNA whose sequence is complementary to the CRISPR guide, the Cas9 (CRISPR-associated protein 9) nuclease is recruited to specifically cleave the invading DNA, resulting in its degradation.
This CRISPR-Cas9 system has been modified for use in mammalian cells. By introducing a guide sequence (sgRNA) specific for our gene of interest, we can either knock-out specific genes through introducing frame shift mutations via Non-Homologous End Joining (NHEJ), or generate knock-in mutations through additionally providing a template for Homologous Recombination (HR).
With BPS Bioscience’s custom cell line development services, we can generate custom knock-out and knock-in cell lines using CRISPR/Cas9 licensed technology. The development process is comprised of five milestones where data is provided after each milestone completion. Each project is customized for the desired deliverables through working directly with our team of highly trained scientists. Contact us to learn more about these services.
BPS offers over 70 cell types across cancerous and noncancerous cell lines. To create a cell line in a different cell type, contact us.
1. Molecular Biology
BPS will design and synthesize three short guide RNA (sgRNA) sequences corresponding to your gene of interest to be knocked-out, based on criteria to maximize cutting efficiency while minimizing potential off-targeting. If you are looking to knock-in a point-mutation or add a tag to an endogenously expressed gene, BPS will also design and synthesize the homology driven repair (HDR) template.
2. CRISPR Transfection
Depending on the cell-type, cells can be transfected via electroporation, liposome-based transfection, or viral infection. The parental cell line can be provided or it will be supplied by BPS.
3. Limiting Dilution
Based upon the results of the initial pool testing, the cell pool will be clonally diluted and the single cell-derived clones will be expanded.
4. Screen For Either Loss of Expression or the Knock-In Mutation
The expression level of the gene of interest will be analyzed via Western Blot or FACS.
Genes showing loss of expression of the gene of interest will be further analyzed through genomic sequencing. Furthermore, for Knock-in mutations, functional validation is available depending on the gene of interest. Mycoplasma testing and cell banking services are also available.