EnGen® Spy Cas9 Nickase is a variant of Cas9 nuclease differing by a point mutation (D10A) in the RuvC nuclease domain, which enables it to nick, but not cleave, DNA (1,2). EnGen Spy Cas9 Nickase, like EnGen Cas9 NLS (NEB #M0652), targets DNA using a guide RNA complementary to a site with a 3’ NGG protospacer adjacent motif (PAM). Nicking occurs on the DNA strand opposite the PAM. Double-stranded DNA breaks can be generated with reduced off-target cleavage by targeting two sites in close proximity (generally 0-20 bp apart) and with PAMs facing outward to leave 5’ overhangs. Nicking by EnGen Spy Cas9 Nickase occurs on the DNA strand opposite the target sequence. EnGen Spy Cas9 Nickase contains SV40 T antigen nuclear localization (NLS) sequences on the N and C- termini.

Product Source
EnGen® Spy Cas9 Nickase is expressed as a N-terminal 6XHis-tagged fusion in E. coli.

  Paired, offset cleavage of genomic loci

  Target-specific DNA nicking

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

1. 20 µM is equal to ~3.2 mg/ml EnGen® Cas9 Nickase NLS, S. pyogenes. 1 µM is equal to ~0.16 mg/ml.

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.  Mali, P., et. al. (2013). NatBiotech. 31 (9), 838-8.

2.  Ran, FA., et. al. (2013). Cell. 154 (6), 1380-9.