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).
- Cas9 Nuclease, S. pyogenes (NEB #M0386 )
- NEBuffer 3.1
- 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
- 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 3.1 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.
- Assemble the reaction at room temperature in the following order:
- Mix thoroughly and pulse-spin in a microfuge.
- Incubate at 37°C for 15 minutes.
- Add 1 μl of Proteinase K to each sample, Mix thoroughly and pulse-spin in a microfuge.
- Incubate at room temperature for 10 minutes.
- Proceed with fragment analysis.
|Component||30 µl reaction|
|Nuclease-free water||20 µl|
|NEBuffer 3.1||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|
- Jinek et al. (2012) Science 337 (6096) 816-821.
- Larson et al. (2013) Nature Protocol 8 (2180-2196).
- Mali et al. (2013) Science 339 (6121): 823-826.