In vitro digestion of DNA with EnGen Spy Cas9 NLS (M0646)
Overview
EnGen Spy Cas9 NLS is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. EnGen Cas9 NLS, S. pyogenes contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the N- and C- termini of the protein. This protocol describes how to digest double-stranded DNA in vitro using EnGen Spy Cas9 NLS and a single guide RNA (sgRNA).
Required Materials:
- EnGen Spy Cas9 NLS (NEB #M0646)
- 10X NEBuffer r3.1
- 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) or using the EnGen® sgRNA Synthesis Kit, S. pyogenes (NEB #E3322S)
- sgRNAs must contain sequence complementary to the target DNA
- 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 EnGen Spy Cas9 NLS 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.
- Prepare 1 µM EnGen Spy Cas9 NLS by diluting the enzyme stock (M0646Tor M0646M) with Diluent B (NEB #B8002S).
Procedure
- 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 analysis.
Component |
30 µl reaction |
Nuclease-free water |
20 µl |
10XNEBuffer r3.1 |
3 µl |
300nM sgRNA |
3 µl (30 nM final) |
1 µM EnGen Spy Cas9 NLS |
1 µl (30 nM final) |
Reaction volume |
27 µl |
Pre-incubate for 10 minutes at 25⁰C |
|
30nM substrate DNA |
3 µl (3 nM final) |
Total reaction volume |
30 µl |
*The substrate DNA and sgRNA, and nuclease-free water are not included.