Cas9 nuclease in the CRISPR-Cas9 system

The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that requires a guide RNA for genomic DNA target recognition and cleavage. All of our Cas9 nuclease products are a human codon-optimized version of the Streptococcus pyogenes Type II Cas9 nuclease.

Fluorescent Cas9 reagents for optimization and enrichment

Enrichment of edited cells in a population is greatly simplified with Cas9 fluorescent lentiviral or mRNA reagents that co-express either mKate2 or TurboGFP™ with Cas9. These reagents also allow for straightforward transfection optimization and visualization of successful Cas9 delivery.

Transient Cas9 products enable a completely DNA-free knockout experiment

Cas9 nuclease mRNA and protein are DNA-free options that permit transient expression of Cas9 to reduce off-targets and protect cells from potential unwanted cellular responses from long term expression. Fluorescent Cas9 mRNA allows for sorting, enrichment and visualization of Cas9 delivery.

Choose from a variety of Cas9 vectors for optimal expression in your system

Promoter activity and resultant Cas9 expression, can vary with cell type; therefore vector-based Cas9 nuclease products are offered with six different, well-characterized, constitutive cellular promoters so you can choose an optimal promoter for your cells (Table 1). Cas9 nuclease is also available with an inducible promoter, permitting you to control the timing of the expression of Cas9, which may be a critical component to a successful experiment.

Table 1. Six SMARTchoice promoter options for expressing Cas9 nuclease

Promoter	Description
hCMV	human cytomegalovirus immediate early promoter
mCMV	mouse cytomegalovirus immediate early promoter
hEF1α	human elongation factor 1 alpha promoter
mEF1α	mouse elongation factor 1 alpha promoter
PGK	mouse phosphoglycerate kinase promoter
CAG	chicken beta actin hybrid promoter
TRE3G	doxycycline inducible promoter

Cas9 nuclease selection guide

Cas9 nuclease formats are not one-size-fits-all. Determining the most appropriate Cas9 nuclease reagent for your experiment is dependent on the particular application or cell type. See the table below to determine the best format for your experiment.

	Cas9 protein	Cas9 mRNA	Cas9 expression plasmid	Lentiviral Cas9 plasmid	Lentiviral Cas9 particles
DNA-free, transient expression	✔	✔
Co-electroporate with synthetic guide RNA	✔	✔
Co-transfect with synthetic guide RNA	✔	✔	✔	✔
Enrich population with FACS		✔	✔	✔	✔
Enrich population with resistance marker			✔	✔	✔
Inducible expression				✔	✔
Create stable cell lines					✔
Lentiviral transduction for cells that are difficult to transfect					✔

Supporting data

Promoter strength driving Cas9 expression correlates with cleavage efficiency

  

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.

Cas9 mRNA Nuclease gene editing in three cell lines

 

HEK293T, A549 and U2OS cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmaFECT 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 Design Tool. UT = untreated sample.

Editing of PPIB, DNMT3B and PSMD11 genes in human cell lines using Dharmacon predesigned crRNAs

 

Efficient gene editing with 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 DharmaFECT 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

Gene editing activity of inducible Cas9 vectors after induction with Doxycycline for 7 days

 

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.

Synthetic crRNA:tracrRNA is compatible with all Cas9 Nuclease formats

 

U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Cas9 Nuclease plasmid (200 ng), 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 µL/well) in biological triplicates.