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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 )
- 10X Cas9 Nuclease Reaction Buffer
- 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) 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 Bioanlyzer 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.
- Assemble the reaction at room temperature in the following order:
*The substrate DNA and sgRNA, and nuclease-free water are not included.
||30 µl reaction
|10X Cas9 Nuclease Reaction Buffer
||3 µl (30 nM final)
|1 µM Cas9 Nuclease, S. pyogenes (M0386S)
||1 µl (~30 nM final)
|Pre-incubate for 10 minutes at 25⁰C
|30nM substrate DNA
||3 µl (3 nM final)
|Total reaction volume
- Mix thoroughly and pulse-spin in a microfuge.
- Incubate at 37°C for 15 minutes.
- Add 1 ul 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.
- 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.