In vitro digestion of DNA with EnGen® Spy Cas9 HF1 (NEB #M0667)


EnGen Spy Cas9 HF1 is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using EnGen Spy Cas9 HF1 and a single guide RNA (sgRNA). 

Required Materials:

  • EnGen Spy Cas9 HF1 (NEB #M0667)
  • 10X NEBuffer r3.1
  • 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) or using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S)
    • sgRNAs must contain sequence complementary to the target DNA
    • 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 EnGen Spy Cas9 HF1 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.
  • Prepare 1 µM EnGen Spy Cas9 HF1 by diluting the enzyme stock (M0667T or M0667M) with 1X NEBuffer r3.1 (NEB # B6003S) for immediate use. For storage at -20°C, dilute the enzyme stock with Diluent B (NEB #B8002S).


  1. Assemble the reaction at room temperature in the following order:

  2. Component

    30 µl reaction

    Nuclease-free water

    20 µl

    10XNEBuffer r3.1

    3 µl

    300nM sgRNA

    3 µl (30 nM final)

    1 µM EnGen Spy Cas9 HF1

    1 µl (30 nM final)

    Reaction volume

    27 µl

    Pre-incubate for 10 minutes at 25⁰C

    30nM substrate DNA

    3 µl (3 nM final)

    Total reaction volume

    30 µl

    *The substrate DNA, 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.