In vitro digestion of DNA with EnGen Spy Cas9 Nickase (M0650)

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). It is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to nick double-stranded DNA in vitro using EnGen Spy Cas9 Nickase and a single guide RNA (sgRNA).

Required Materials:
  • EnGen Spy Cas9 Nickase (NEB #M0650)
  • 10X NEBuffer r3.1 Reaction Buffer
  • Nuclease-free water
  • Proteinase K, Molecular Biology Grade (NEB #P8107)
  • sgRNA containing the targeting sequence in the region of interest
    • sgRNAs can be generated by in vitro transcription using the HiScribe T7 Quick High-Yield RNA synthesis Kit (NEB #E2050) using linearized plasmid, PCR products, or oligonucleotides as templates
    • sgRNAs must contain sequence complementary to the target DNA (3,4)
    • For information on design of sgRNA transcription templates please visit Addgene
  • DNA substrate containing the target sequence
  • The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides
Optional Materials:
  • Apparatus and reagents for DNA fragment analysis
    • E.g. Agarose gel electrophoresis apparatus
      • DNA Loading Dye (e.g. Gel Loading Dye, Purple (6X) (NEB #B7024S)
    • E.g. Agilent Bioanalyzer or similar
Before You Start:
  • We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found here.
  • Reactions are typically 30 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
  • It is essential to keep the molar ratio of Cas9 Nickase and sgRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here.
  • Prepare 300 nM sgRNA by diluting the stock with nuclease-free water on ice.
  • Prepare 30 nM substrate DNA with a single target sequence by diluting the stock with nuclease-free water on ice.
  • If planning to use higher concentration EnGen Spy Cas9 Nickase (NEB #M0650T) for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X NEBuffer r3.1 prior to the reaction assembly and used immediately. If the 1 μM dilution will be stored at -20°C, it should be diluted using Diluent B (NEB #B8002S): 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. Assemble the reaction at room temperate in the following order:
    30 μL REACTION
     Nuclease-free water
    20 μl
    10X NEBuffer r3.1 Reaction Buffer
    3 μl
    300 nM sgRNA
    3 μl (30 nM final)
    1 μM EnGen Spy Cas9 Nickase (M0650)
    1 μl (~30 nM final)
    Reaction volume
    27 μl
     Pre-incubate for 10 minutes at 25°C
    30 nM substrate DNA
    3 μl (3 nM final)
    Total reaction volume
    30 μl
    * The substrate DNA and sgRNA, and nuclease-free water are not included.

  3. Mix thoroughly and pulse-spin in a microfuge.
  4. Incubate at 37°C for 15 minutes.
  5. Add 1 μl of Proteinase K to each sample. Mix thoroughly and pulse-spin in a microfuge.
  6. Incubate at room temperature for 10 minutes.
  7. Proceed with analysis.
  1. Mali, P., (2013) Nat Biotech 31 (9), 838-8.
  2. Ran, FA., (2013) Cell 154 (6), 1380-9.
  3. Jinek et al. (2012) Science 337 (6096) 816-821.
  4. Larson et al. (2013) Natural Protocol 8 (2180-2196).