In vitro digestion of plasmid DNA with EnGen SpRY Cas9 (NEB #M0669)


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

Required Materials

  • EnGen SpRY Cas9 (NEB #M0669)
  • 10X NEBuffer r3.1
  • Nuclease-free water
  • 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 the design of sgRNA transcription templates, please visit Addgene
  • Plasmid DNA containing the target sequence

Optional Materials

  • Apparatus and reagents for DNA fragment analysis
    • Agarose gel electrophoresis apparatus
      • DNA Loading Dye (e.g. Gel Loading Dye, Purple (6X) (NEB #B7024S))
    • 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 50 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
  • We recommend 1 µM final concentrations of EnGen SpRY Cas9 and sgRNA per 1 µg of plasmid for best cleavage efficiency. However, we have also observed high-efficiency cleavage of 1 µg of plasmid with SpRY Cas9 and sgRNA concentrations as low as 50 nM.
  • Prepare 20 µM sgRNA by diluting the stock with nuclease-free water on ice.
  • Prepare 500 ng/µl plasmid DNA by diluting the stock with nuclease-free water on ice.
  • If using lower concentrations of EnGen SpRY Cas9, a working solution may be prepared by diluting the 20 µM enzyme stock (NEB #M0669T or #M0669M) 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:


    50 µl reaction

    Nuclease-free water

    38 µl

    10X NEBuffer r3.1

    5 µl

    20 µM sgRNA

    2.5 µl (1 µM final)

    20 µM EnGen SpRY Cas9

    2.5 µl (1 µM final)

    Reaction volume

    48 µl

    Pre-incubate for 10 minutes at 25⁰C

    500 ng/µl plasmid DNA

    2 µl (1 µg final)

    Total reaction volume

    50 µl

    *The plasmid DNA, sgRNA, and nuclease-free water are not included.

  2. Mix thoroughly and pulse-spin in a microfuge.

  3. Incubate at 37°C for 1 hour.

  4. Incubate at 80°C for 5 minutes to heat inactivate the enzyme.

  5. For optimal results, clean up the linearized plasmid using Monarch PCR & DNA Cleanup Kit (NEB #T1030S or #T1030L).|

  6. Proceed with the desired cloning workflow. For NEBuilder HiFi DNA Assembly (NEB #E2621, #E2623, or #E5520), follow the recommended instructions.