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Configurable expression constructs or DNA-free options for optimal Cas9 expression
Active expression of the Cas9 endonuclease is a critical requirement for efficient gene editing. The Edit-R portfolio of Cas9 nuclease expression vectors includes six SMARTchoice constitutive promoter options or an inducible format, providing the flexibility to select an optimal system for robust expression and maximal cleavage efficiency when Edit-R guide RNAs are introduced into the cell.
In type II CRISPR-Cas systems, the CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that requires a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) for genomic DNA target recognition and cleavage using two active sites that together generate DNA double-strand breaks (DSB).
The Edit-R Cas9 nuclease expression vectors incorporate a human codon-optimized version of the Streptococcus pyogenes Type II Cas9 (formerly csn1) gene. The activity of any given promoter controlling the transcription of Cas9 nuclease can differ greatly from one biological context to another, resulting in variable Cas9 expression levels and thus varying levels of DNA cleavage. Vector-based Edit-R Cas9 Nuclease products are offered with up to six different, well-characterized constitutive cellular promoters so you can choose an optimal promoter for your cells (Table 1). Edit-R Cas9 Nuclease is also available with an inducible promoter format, thus permitting you to control the timing of the expression of Cas9, which may be a critical component to a successful experiment.
DNA-free Cas9 nuclease options protect against long-term expression of Cas9 nuclease when transient expression is desired. With Cas9 mRNA and Cas9 protein there is also no risk of plasmid integration into the host genome or side-effects from constitutive Cas9 expression.
Non-lentiviral vectors provided as endotoxin-free purified DNA for direct co-transfection with Edit-R synthetic crRNA and tracrRNA.
Purified lentiviral particles or plasmid DNA for generation of stable Cas9 nuclease-expressing cell populations. Constitutive or inducible promoter options are available.
Purified Cas9 mRNA for transient Cas9 Nuclease expression.
Purified Cas9 protein ready to use for DNA-free nuclease expression.
While the best Cas9 nuclease product for your experiment may heavily depend on the particular application or cell type, a few basic questions may help to point you in the right direction for product selection.
Differential Cas9 expression driven by multiple promoters correlate with cleavage efficiency of several target genes in HEK293T cells. Cells were transfected with SMARTCas9-mKate2 expression plasmids with six different promoters, tracrRNA and crRNAs targeting human DNMT3B, PPIB, CDKN1A or VCP genes. Top panel: Images of cells expressing mKate2 fluorescent reporter were taken 48 hours post-transfection. Mouse EF1α promoter shows strongest expression of the mKate2 reporter. Bottom panel: Without enrichment (i.e., no cell sorting), HEK293T cells were lysed at 72 hours post-transfection and PCR was performed with primers flanking the cleavage site. SURVEYOR™ DNA mismatch assay was performed and the samples were separated on a 2% agarose gel. The level of editing was calculated using densitometry (% editing). UT = Untransfected. The mouse EF1α promoter provides the highest mKate2 expression which correlates with higher % gene editing in HEK293T cells.
HEK293T, A549 and U2OS cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmFECT Duo Transfection Reagent with Cas9 mRNA (200 or 100 ng) and synthetic crRNA:tracrRNA (50 nM) targeting VEGFA. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. The synthetic crRNA was designed using the Dharmacon CRISPR RNA Configurator. UT = untreated sample.
Efficient gene editing with Edit-R Cas9 Nuclease protein NLS demonstrated by DNA mismatch assay using T7 Endonuclease I. U2OS and HeLa cells were plated at 10,000 cells/well in 96-well plates and co-transfected using DharmFECT 1 Transfection Reagent with Cas9 Nuclease protein (25 nM) and synthetic crRNA:tracrRNA (50 nM) targeting PPIB, DNMT3B and PSMD11. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. UT = untreated sample, NTC = non-targeting control
Cells were transduced with a constitutive (CAG-Cas9) or an inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with DNMT3B- or PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin for 2 days in tetracycline-free medium and split in two populations: uninduced (DOX-) and induced (DOX+) with 500 ng/mL doxycycline for 7 days. The cells were then lysed and analyzed for indels using a DNA mismatch detection assay with T7EI.
U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R Cas9 Nuclease plasmid (200 ng), Edit-R Cas9 Nuclease mRNA(200 ng) or Cas9 nuclease protein (25 nM) and crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT Duo transfection reagent (0.4 L/well) in biological triplicates.