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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: Hek293 cell culture transfected with a construct including the C terminal 814-1372 amino acids of Staphylococcus pyogenes Cas9. The cells were stained with RA22126 in red and also with a chicken antibody to S. pyogenes Cas9 in green. A cell expressing Cas9 therefore appears yellow. Most Hek293 cells are not transfected so only the nucleus of these cells can be visualized with the blue 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 an affinity purified polyclonal raised in rabbit against a mixture of two recombinant constructs corresponding to amino acids 1-608 and 814-1372 of Cas9 from the Streptococcus pyogenes and binds both these proteins transfected into cells and on western blots, and also recognizes the full length protein. 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.
Image: Western blot analysis of RA22126
Blot of 100 ng (lane1) and 10 ng (lane2) of full length Cas9 protein from Streptococcus pyogenes was probed with RA22126 at 1:2,000 dilution. This antibody recognizes full length Cas9 protein at 160 kDa and a proteolytic fragment at 130 kDa.