The development of the CRISPR-Cas technology allows scientists to edit genomes relatively quickly and relatively easily, providing a sophisticated, powerful tool to introduce a variety of genetic alterations (e.g. deletions, insertions, base pair mutations) into mammalians cells. This approach involves a synthetic, programmable guide RNA (gRNA) that is associated with a Cas9 nuclease. This tailored single guide RNA (sgRNA) can bind to a specific dsDNA sequence, bringing the CAS9 nuclease to a targeted region in the genome, where the nuclease then introduces a double-strand break in the DNA. By using the cell’s endogenous DNA repair mechanisms, it is possible to engineer very specific genomic changes.
A crucial step of any CRISPR workflow is delivering the sgRNA and Cas9 protein into the cytoplasm or nucleus of the target cell. Guide-RNA and Cas9 can be delivered into cells as (plasmid) DNA, mRNA, or as pre-formed ribonucleoprotein complexes (RNPs).
Delivering the CRISPR-Cas constructs into hard-to-transfect cell types such as primary cells, stem cells or neurons can be quite challenging. Various methods have been developed to efficiently introduce nucleic acids or RNPs into primary cells and cell lines. This must be done while maintaining cell heath and morphology, along with avoiding off-target effects.
The PromoCell PromoFectin family of transfection reagents provides complete delivery solutions for successful CRISPR/Cas9 genome editing. Our high-quality reagents and optimized protocols for each product option will help you significantly improve your genome editing results with multiple – even difficult-to-transfect – cell types. In contrast to harsh, expensive but frequently used methods such as electroporation, our economic PromoFectins mediate a gentle transfection with lowest cytotoxicity.
CRISPR-Cas component delivery options:
Plasmids carrying the coding sequences for the gRNA and Cas9 enter the nucleus and are transcribed into RNA (gRNA or mRNA that will be translated into Cas9) that enter the cytoplasm. Use:
The Cas9-encoding mRNA will be directly delivered into the cytoplasm and then translated. Use:
Ribonucleoprotein complex (RNP)
Pre-complexed RNPs do not require any transcription or translation and are delivered directly to the cytoplasm or to the nucleus. Use:
To learn more about PromoCell’s PromoFectin Transfection Reagents, visit our webpage , or get in touch with our scientific support service at the toll-free number 0800 -776 66 23.