Rabbit Polyclonal Antibody to S. aureus CAS9 Cat# RPCA-CAS9-Sa

      A recent revolution in biology has been stimulated by the discovery of CRISPR, or “Clustered Regularly Interspaced Short Palindromic Repeats” and the understanding of the "CRISPR Associated" enzymes (CAS 1,2). The CRISPR repeated sequences are found in bacterial genomes and function as part of unique bacterial immune system which contain 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 CAS9 is directed to cleave the specific viral sequence and so inactivate the virus. The RNA sequence can be designed to specifically cut DNA virtually anywhere, including in the genomes of living human and other mammalian cells, allowing inexpensive gene editing with unprecedented ease. For example three groups of researchers essentially cured the disease state in a mouse model of Duchenne muscular dystrophy (3-5). A similar approach essentially cured dogs affected with a related disease state (6). Several varieties of CAS9 have been studied and there are several other related enzymes with similar properties. Much of the early work was performed with CAS9 from Streptococcus pyogenes which is rather large at ~158kDa, so the corresponding DNA is also rather large at about 4.2kb. This is problematic with some expression systems especially since DNA encoding RNA sequences and possibly other regulatory elements are usually required. The CAS9 gene of Staphylococcus aureus is significantly smaller, 3kb, producing a protein of 124kDa (6). For an excellent recent review of the various CAS family enzymes and their utility see reference 8.

The antibody has not been tested on formalin fixed paraffin embedded sections, and so we cannot recommend it for this purpose.

The S. aureus Cas9 sequence was based on that was GenBank CCK74173.1 which was inserted into the eukaryotic expression vector pET30a(+) which adds an N-terminal His-tag and some other sequence, underlined below. This sequence includes a thrombin cleavage site (blue), an S-tag affinity peptide (red) and an enterokinase cleavage site (green).

MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMADIGS EFNNLNGLYD
KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK
KDNGPVIKKI KYYGNKLNAH LDITDDYPNS RNKVVKLSLK PYRFDVYLDN GVYKFVTVKN
LDVIKKENYY EVNSKCYEEA KKLKKISNQA EFIASFYNND LIKINGELYR VIGVNNDLLN
RIEVNMIDIT YREYLENMND KRPPRIIKTI ASKTQSIKKY STDILGNLYE VKSKKHPQII
KKG

Number of amino acids: 303
Molecular weight: 35419.39
Theoretical pI: 9.16

Amino acid composition:
Ala (A) 10 3.3%
Arg (R) 9 3.0%
Asn (N) 27 8.9%
Asp (D) 24 7.9%
Cys (C) 1 0.3%
Gln (Q) 7 2.3%
Glu (E) 18 5.9%
Gly (G) 15 5.0%
His (H) 11 3.6%
Ile (I) 22 7.3%
Leu (L) 26 8.6%
Lys (K) 43 14.2%
Met (M) 8 2.6%
Phe (F) 6 2.0%
Pro (P) 11 3.6%
Ser (S) 16 5.3%
Thr (T) 11 3.6%
Trp (W) 0 0.0%
Tyr (Y) 24 7.9%
Val (V) 14 4.6%

Total number of negatively charged residues (Asp + Glu): 42
Total number of positively charged residues (Arg + Lys): 52

This protein does not contain any Trp residues. Experience shows that
this could result in more than 10% error in the computed extinction coefficient.

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 35760