EnGen® Spy dCas9 is an inactive mutant of Cas9 nuclease that retains programmable DNA binding activity (1,2). EnGen Spy dCas9 contains two point mutations, D10A and H840A, which inactivate its RuvC and HNH nuclease domains, respectively. The N-terminal SNAP-tag® allows for covalent attachment of fluorophores, biotin, and a number of other conjugates useful for visualization and target enrichment. Like with Cas9 nuclease, DNA binding by EnGen Spy dCas9 requires a guide RNA complementary to a target sequence with a protospacer adjacent motif (PAM), NGG, downstream of the target sequence. EnGen Spy dCas9 contains a SV40 T antigen nuclear localization sequence (NLS) at the C- terminus of the protein.

Product Source

EnGen® Spy dCas9 is expressed as a N-terminal 6XHis-tagged fusion in E. coli.

  Localization of DNA targets in vivo

  Target Enrichment

Reaction Conditions

1X NEBuffer 3.1.  Incubate at 37°C.

1X NEBuffer™ 3.1:
100 mM NaCl
50 mM Tris-HCl
10 mM MgCl₂
100 μg/ml BSA
pH 7.9 @ 25°C
 

Storage Temperature

-20°C

Storage Conditions

300 mM NaCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C

Companion Products

• EnGen® sgRNA Synthesis Kit, S. pyogenes
• HiScribe™ T7 Quick High Yield RNA Synthesis Kit
• SNAP-Vista® Green
• SNAP-Capture Magnetic Beads

1.   20 µM is equal to ~3.6 mg/ml and 1uM is equal to ~ 0.18 mg/ml EnGen® Spy dCas9.

2.   This product is compatible with Diluent Buffer B: 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA,

     1 mM DTT, 500 µg/ml BSA and 50% glycerol. (pH 7.4 @ 25°C).

1.  Qi, LS., et. al. (2013). Cell. 152 (5), 1173-83.

2.  Qi, LS., et. al. (2013). Cell. 154 (2), 442-51.