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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 overexpressing the N-terminal 1-608 amino acids of S. pyogenes Cas9 . The cells were stained with MO22174 in red, and these cells also appear yellow since we counterstained with our rabbit Cas9 antibody, RA22126 to the same construct in green, giving a yellow color. The N-terminal construct contains a nuclear localization sequence and so is predominantly nuclear in localization. Most Hek293 cells in this field are not transfected so only the nuclei of these cells can be visualized with the blue DAPI DNA stain.
Several varieties of Cas9 have been studied and there appear to be several other related enzymes with similar properties in bacteria. Two Cas9 homologs include Streptococcus pyogense and Staphylococcus aureus. Staphylococcus aureus is significantly smaller and so presents less problems when packaged into vectors. 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. Our antibody is a mouse monoclonal raised against amino acids 1-608 of Cas9 from S. pyogenes and binds the immunogen transfected into cells on western blots and in immunocytochemistry. The homologous region of the S. aureus Cas9 is not closely related in amino acid sequence and, as expected, this antibody does not recognize that protein.
Western blot analysis of MO22174
Lane 1: Protein size marker with size in kiloDaltons..
Lane 2: Blot of crude protein extract from Hek293 culture transfected with MO22174 immunogen, the N-terminal 1-608 amino acids.
Lane 3: Non-transfected control Hek293 cell extract.
Lane 4: Blot of 40 ng Streptococcus pyogenes Cas9 was probed with MO22174 at 1:1,000 dilution and, as expected, the antibody also recognizes full length Cas9 protein at 160 kDa.