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The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has changed the field of gene editing. These repeated sequences are found in bacterial genomes with short DNA sequences derived from viruses which have infected the bacteria interspaced. 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 are 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 enzymes and their relatives for use in mammalian systems.
Image: Transfected HEK293 cells with GFP-Cas9-SA fusion protein were stained with MO22175 antibody. GFP is expressed in the transfected cells (green). GFP-Cas9 fusion protein is stained with MO22157 antibody at 1:1,000 dilution (red). Only transfected cells are reactive with MO22157 antibody, which appear in a yellow-orange color (merge). Nuclei are visualized in blue with Hoechst staining.
Several varieties of Cas9 have been studied and there appear to be several o ther related enzymes with similar properties in bacteria. Two Cas9 homologs include Streptococcus pyogense and Staphylococcus aureus. 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. Staphylococcus aureus is significantly smaller, at about 3 kb producing a protein 124 kDa. Thus presents less problems when packaged into vectors. Our antibody is a mouse monoclonal raised 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
Image: Western blot analysis of MO22175
1: HEK293 cells which overexpressed fusion protein containing GFP and c-terminus of Cas9 from S. aureus.
2: Non-transfected HEK293 cells.
There is a strong clean band at about 53 kDa corresponding to GFP-Cas9 fusion protein, which is absent in non-transfected cells. MCA-6F7 antibody is diluted at 1: 1,000 dilution