For the standard CRISPR system, knockout efficiency is directly dependent on Cas9 expression levels. Cell lines stably expressing Cas9 are required for CRISPR screening with pooled sgRNA libraries. Transient expression of the Cas9 is not sufficient for pooled screening. For CRISPR screening purposes, it is important to use cells that express Cas9 well. Further, Cas9 expression levels definitely vary from cell type to cell type, depending on the promoter used to drive Cas9 expression and the number of Cas9 copies per cell. And, to ensure optimal expression in your cell model for screening and gene editing, Cellecta offers a range of different Cas9 and dCas9-variant expressing constructs.
For the standard CRISPR system, knockout efficiency is directly dependent on Cas9 expression levels and transient expression is not reliable for genetic screens with pooled sgRNA libraries. For CRISPR screening purposes, it is important to use cells that express Cas9 well. Further, Cas9 expression levels definitely vary from cell type to cell type, depending on the promoter used to drive Cas9 expression and the number of Cas9 copies per cell. To ensure optimal expression in your cell model for screening and gene editing, Cellecta offers a range of different Cas9 and dCas9-variant expressing constructs.
The sgRNA contains an initial variable region of about 20 bases, which targets the DNA region of interest, followed by a constant sequence of about 80 nucleotides that contains all the key interactions with the Cas9 nuclease. In the original CRISPR knockout system, the sgRNA/Cas9 nuclease complex effectively repeatedly cleaves the genomic DNA at the targeted target site until the repair process fails and produces an insertion or deletion (i.e, an “indel”) that typically knocks out gene expression.
CRISPRa and CRISPRi systems use a deactivated Cas9 gene (dCas9) fused with either a gene repressor (i.e., the KRAB protein) or a complex of transcription activators to create engineered dCas9 variants that, when targeted to a promoter region by an sgRNA, modulates transcription activation or repression of the target gene. This approach does not alter the underlying DNA structure, like a CRISPR knockout does, and so the induction or repression is reversible.
The genes for all the Cas9 variations are over 4kb in size and thus are relatively large for lentiviral constructs. As a result, packaging of the Cas9 and dCas9 constructs is somewhat inefficient. It is for this reason that Cellecta's Cas9 constructs contain only one selection marker--either an antibiotic resistant gene or a fluorescent protein. Since the the expression level of the relatively large Cas9 protein can vary with different promoters in different cell systems and experiments, it is important to select a Cas9 expression construct with the best features for your cell model and experimental plan.
