Cas9 from S. pyogenes

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.

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.