In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 


Cas9 Nuclease, S. pyogenes, (Cas9) 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 Cas9 and a single guide RNA (sgRNA).

Required Materials:

  • Cas9 Nuclease, S. pyogenes (NEB #M0386 )
  • NEBuffer r3.1
  • Nuclease-free water
  • Proteinase K, Molecular Biology Grade (NEB #P8107S)
  • sgRNA containing the targeting sequence in the region of interest
    • sgRNAs can be generated by in vitro transcription using the EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322) and a single oligonucleotide or 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 (1,2)
    • 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 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 Cas9 Nuclease, S. pyogenes (NEB #M0386T and NEB #M0386M) for in vitro digestion of DNA, the enzyme can be diluted to 1 µM in 1X Buffer r3.1 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) prior to the reaction assembly.


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

  2. Component 30 µl reaction
    Nuclease-free water 20 µl
    NEBuffer r3.1 3 µl
    300 nM sgRNA 3 µl (30 nM final)
    1 µM Cas9 Nuclease, S. pyogenes (M0386S) 1 µl (~30 nM final)
    Reaction volume 27 µl
    Pre-incubate for 10 minutes at 25⁰C
    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 fragment analysis.


  1. Jinek et al. (2012) Science 337 (6096) 816-821.
  2. Larson et al. (2013) Nature Protocol 8 (2180-2196).
  3. Mali et al. (2013) Science 339 (6121): 823-826.