Cas9 from Staphylococcus aureus , rabbit polyclonal, cat#RPCA-Cas9-SA,

Cas9 from Staphylococcus aureus , rabbit polyclonal, cat#RPCA-Cas9-SA,



Transfected HEK293 cells overexpressing a GFP-Cas9-SA fusion protein were stained with RPCA-Cas9-SA. Cells which are transfected with GFP-Cas9 are bright green (left panel). Staining with RPCA-Cas9-SA is shown in red in middle panel. In merged image (right panel), most HEK293 cells are not transfected so only the nucleus of these cells can be visualized with a blue DNA stain. Red antibody staining is only seen in cells which express GFP, as expected, and the superimposition of green and red results in an orange signal.

Western blot analysis of RPCA-Cas9-SA
1: HEK293 cells overexpressing a fusion protein containing GFP and the C-terminus of Cas9 from S. aureus.
2: Non-transfected HEK293 cells.The band at about 53 kDa corresponds to the GFP-Cas9 fusion protein and it is absent from non-transfected cells.
RPCA-Cas9-SA is at 1: 2,000 dilution

Product name Anti-Staphylococcus aureus Cas9
Description Rabbit polyclonal to the C-terminal region of Cas9 from Staphylococcus aureus
Reference Code RPCA-Cas9-SA
HGNC name  NA, no human homolog
RRID# AB_2572246
Molecular weight 124kDa
Immunogen C-terminal region of S. aureus, amino acids 803-1053 of sequence CCK74173, expressed in and purified from E. coli.
Isotype IgG
Concentration Antibody is supplied as 100µL rabbit crude serum
Applications Western blot, ICC/IF,
Suggestions for use Western blot: 1:1,000-1:5000. ICC/IF:1:5,000 .
Storage instructions Shipped on ice. Store at 4°C. For long term storage, leave frozen at -20°C. Avoid freeze / thaw cycles.


A recent revolution is biology has been stimulated by the discovery of CRISPR, or “Clustered Regularly Interspaced Short Palindromic Repeats” and the understanding of their significance. These repeated sequences are found in bacterial genomes and function as part of unique bacterial immune system. Interspaced between these repeated DNA sequences are short DNA sequences derived from viruses which have infected the bacteria. These virally derived sequences can make short RNA sequences which can hybridize with specific viral DNA and target a nuclease, such as Cas9, to the viral sequence. So, if the bacteria is infected by this virus again, Cas9 can be directed to cleave the specific viral sequence and so inactivate the virus. By careful design of the RNA sequence the system can be used to specifically cut DNA virtually anywhere, including in living human and other mammalian cells. This allows inexpensive gene editing with unprecedented ease, and much effort is going into refining the Cas9 for use in mammalian systems. Recent papers in this exploding field showed that it is feasible to correct genetic defects in a variety of experimental situations. For example three groups of researchers essentially cured the disease state in a mouse model of Duchenne muscular dystrophy, a disease in which point mutations or frame shifts result in the production of a truncated and non-functional form of very large muscle protein dystrophin (1). This was performed using AAV vectors on adult animals, using RNA sequences which directed cleavage of the DNA at two sites flanking the genetic defect. The normal DNA repair mechanisms in some cases annealed the two cut sites leaving out the defective region. This allowed the production of a slightly shorter but still functional dystrophin protein. Several varieties of Cas9 have been studied and there appear to be several other related enzymes with similar properties in bacteria. Much of the early work was performed with Cas9 from Streptococcus pyogenes. The S. pyogenes protein is rather large at 1,368 amino acids, ~160kDa, so the corresponding DNA is also rather large at about 4.2 kb. This will not fit easily into some expression systems especially since DNA encoding RNA sequences and possibly other regulatory elements are usually required. In one recent study a group in the Broad Institute searched for the smallest possible Cas9 across known bacterial genomes and found that the version expressed in Staphylococcus aureus was significantly smaller, at about 3 kb, producing a protein of 124kDa (2). Our antibody is a polyclonal raised in rabbit against the C-terminal 251 amino acids of of the Staphylococcus aureus protein and binds this protein transfected into cells on western blots and in immunocytochemistry. The homologous region of the S. pyogenes is not closely related in amino acid sequence and, as expected, this antibody does not recognize that protein.

References:

1. CRISPR helps heal mice with muscular dystrophy.
CRISPR helps heal mice with muscular dystrophy

2. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature 520:186-91 (2015).

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