Cas9 from Streptococcus pyogenes is the most frequently used cas endonuclease in CRISPR. The dCas9 (deficient Cas9 or Dead cas9) is the mutant of spCas9. It lacks the cleavage ability (the mutation removed endonuclease activity), but can still bind to any guide RNAs (gRNA), thus binding to genomic DNA targeted site by gRNA.
What is the application for dCas9?
The dCas9 binds to its DNA target. This binding alone can prevent the cell’s transcription machinery from accessing the binding region (such as the promoter), hence affecting the downstream gene expression. That is the dCas9 can be used in CRISPR interference (CRISPRi) or activation (CRISPRa).
When dCas9 fused with a transcriptional activator, it can activate gene expression without changing DNA sequence (CRISPRa), or repress gene expression by coupling to a transcriptional repressor domain, such as KRAB ), so-called CRISPR interference (CRISPRi). dCas9 can also alter local chromatin structures and make the genomic target sites accessible to other enzymes (such as methyltransferase, or nucleases) for epigenetic changes in gene expression.
Gentarget’s dCas9 products?
Gentarget Inc generated the dCas9 expression construct by making two point mutation in spCas9’s endonuclease domains (RuvC and HNH), as D10A and H840A, resulting in its deactivation. The dCas9 expression is in-framed with an enhanced NLS (nuclear localization signal), and a HA tag (to purify or detect the expressed dCas9 when desired) . The premade dCas9 expression lentivirus contain different selection marker (Antibiotics, Fluorescent, or Antibiotic-Fluorescent fusion dual marker) for transduction verification, cell sorting and selection.
Because each promoter’s strength is cell type dependent, Gentarget made the dCas9 (or dCas9-Enzyme fusion) driven by different promoters, such as enhanced CMV (for many general cell types), enhanced EF1a (for many specific cell types), and SFFV promoter (for HSCs).