Home Protocols In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)

In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)

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Overview:

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.

Procedure:

  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.

  2. Mix thoroughly and pulse-spin in a microfuge.
  3. Incubate at 37°C for 15 minutes.
  4. Add 1 μl of Proteinase K to each sample, Mix thoroughly and pulse-spin in a microfuge. 
  5. Incubate at room temperature for 10 minutes.
  6. Proceed with fragment analysis.


References:

  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.