Hek293 cell culture transfected with a construct including the C terminal 814-1372 amino acids of Staphylococcus pyogenes
Cas9. The cells were stained with RPCA-Cas9-AP 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.
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 a unique bacterial immune system. Interspaced between these repeates 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 the 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 effectively bypassing the defective region. This allowed the production of a slightly shorter but still functional dystrophin protein. There seems to be no reason why this would not work in humans. Several varieties of Cas9 including the significantly smaller homolog from Staphylococcus aureus
have been studied and there are several other bacterial enzymes with similar properties (2). Much of the early work was performed with Cas9 from Streptococcus pyogenes
. 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 regions of the S. aureus
Cas9 are not closely related in amino acid sequence and, as expected, this antibody does not recognize that protein. We recently made available MCA-3F9
, a monoclonal antibody binding to the N-terminal region of Cas9 from S. mutans.
We also have made available a monoclonal antibody MCA-6F7
and polyclonal antibodies RPCA-Cas9-SA made in rabbit and CPCA -Cas9-SA in chicken against Cas9 protein in S. aureus.
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).