Eupheria offers a wide range of different recombinant Streptococcus (S.) pyogenes Cas9 proteins, all of which are highly pure and effective. Fusion with a nuclear localization signal (NLS) ensures targeting of the Cas9 proteins to the nucleus. Successful genome editing by Eupheria Cas9-NLS proteins has been confirmed in a wide variety of mouse and human cell lines, and organisms, including zebrafish, Xenopus and marine plankton.
Try our Cas9-NLS proteins for your CRISPR/Cas9 experiments for greater genome editing specificity, less cell toxicity and higher insertion/deletion (indel) frequencies than with plasmid- or mRNA-encoded Cas9.
Standard scales are 20, 40 and 100 µg. For pricing and other scales, please contact us.
Zebrafish embryos (1- to 2-cell stage) from transgenic parents, expressing EGFP-labeled myosin in the heart, were each injected with 1 ng of Cas9-NLS protein and 100 pg guide RNA targeting EGFP. Forty-eight hours post-injection, no EGFP fluorescence was detectable as a result of the Cas9-NLS-mediated knockout of the EGFP gene. In the control larvae, not injected with Cas9-NLS, the eGFP fluorescence was clearly visible in the two heart chambers.
The Cas9-NLS represents the wild-type Cas9 nuclease from S. pyogenes, fused with a C-terminal NLS. Currently, this the best-established Cas9 protein in the field, which allows you to draw experimental advice from a large body of publications.
This Cas9-NLS protein contains the D10 substitution, which converts it into a nickase. The specific binding of two gRNAs in close proximity on opposite DNA strands is required for the Cas9-Nickase-NLS to cause a double-strand break. Therefore, the Cas9-Nickase-NLS provides the benefit of fewer off-targets compared with the Cas9-NLS nuclease.
The Cas9-Dead-NLS protein is catalytically inactive ("dead") due to the D10A and H840A substitutions. Thus Cas9-Dead-NLS binds to DNA, but does not modify it. This property allows specific genomic targeting, e. g. to block transcription.
As another innovative Cas9 nuclease from Eupheria, the NLS is followed by a C-terminal tag red-fluorescent protein (tagRFP) sequence. The transfection efficiency of Cas9-NLS-tagRFP is as high as for the untagged Cas9-NLS nuclease, but the red fluorescence of successfully transfected cells allows their selection by FACS (fluorescence-activated cell sorting). The genome editing rates of such selected cell populations is 3-4 times higher than in non-selected ones.
555 nm/584 nm
This innovative Cas9 nuclease from Eupheria is composed of a Cas9 sequence from Streptococcus pyogenes followed by a nuclear-localization sequence (NLS) and a C-terminal enhanced green fluorescent protein (EGFP) tag. Similar to Cas9-NLS-tagRFP the transfection efficiency of Cas9-NLS-EGFP protein is as high as for the untagged Cas9-NLS nuclease. The green fluorescence allows following successfully transfected cells by e.g. fluorescence-activated cell sorting (FACS) or fluorescence microscopy. Enriching successfully transfected cells by sorting increases genome editing rates and largely reduces efforts in screening and identification of edited cells.
489 nm/509 nm
This Cas9 protein is catalytically inactive ("dead") due to the D10A and H840A substitutions. The NLS is followed by a C-terminal enhanced green-fluorescence protein (EGFP) sequence. The EGFP-tag offers many application possibilities, e. g. tracking of Cas9-Dead-NLS-EGFP.
488 nm/507 nm
Production: The genes encoding the Cas9 proteins are expressed in E. coli.
NLS: The nuclear localization sequence (NLS) originates from the Simian virus 40.
Quality control: The purity of the Cas9 proteins is checked by Coomassie Blue staining after electrophoresis on SDS-polyacrylamide gels. The expected activity is confirmed in vitro and in cell culture with the mouse embryonic stem cell line E14 and the human osteosarcoma cell line U2OS.
Concentration: The minimum concentration is 500 ng/µL. Upon request, custom concentrations up to 5.0 µg/µL are available.
Storage and Stability: Cas9 proteins stored at -20°C are stable for at least one year. No decrease in activity was observed after 10 freeze/thaw cycles.
Storage Buffer: 20 mM HEPES pH 7.25, 150 mM KCl, 1 mM DTT.
Shipment: Shipped frozen in storage buffer at -20°C.