SpCas9

Alternate Names:

Cas9, Csn1, Spy_1046

UniProt Number:

Q99ZW2

Overview:

Streptococcus pyogenes Cas9 is a type II-A Cas9 protein and is the most used in most current CRISPR-based genetic engineering methodologies (1). CRIPSR RNAs (crRNAs) fused to trans-activating CRISPR RNAs (tracrRNA) direct sequence-specific cleavage by Cas9, introducing double-stranded breaks in cell-invading DNA in bacterial and archaeal CRIPSR/Cas9 systems (1,2). Cas9 requires the 5’-NGG-3’ protospacer adjacent motif (PAM) to destabilize the target DNA (3). Cas9’s HNH endonuclease domain cleaves the strand complementary to the crRNA while its RuvC endonuclease domain cleaves the non-complementary strand (2).

Accession Number:

AAK33936.1

Formulation:

Protein stored in 50mM sodium phosphate, pH 8.0, 300mM NaCl, 150mM imidazole, 1mM DTT, 10% glycerol

References:

1. Jinek, M. et al. 2014. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 343(6176): 1247997.
2. Jinek, M. et al. 2012. A programmable dual RNA-guided endonuclease in adaptive bacterial immunity. Science. 337(6096): 816-821.
3. Anders, C. et al. 2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 513(7519): 569-573.

Storage and Stability:

Store at –70°C. To avoid repeated handling and multiple freeze/thaw cycles aliquot diluted product into smaller quantities.

Concentration:

0.5 μg/μl
6.26 pmol/μg

Purity:

>80%

Recommendation:

Always include RNase inhibitor at recommended concentrations in Cas enzyme reactions

Molecular Weight:

Calculated: 159.7 kDa
Observed: ~150 kDa