In vitro digestion of DNA with EnGen® Lba Cas12a (Cpf1) (M0653)

Overview:

EnGen Lba Cas12a (Cpf1) from Lachnospiraceae bacterium ND2006 is a site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA) (1). Targeting requires a gRNA complementary to the target site as well as a 5´ TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Cleavage by EnGen® Lba Cas12a occurs ~18 bases 3´ of the PAM and leaves 5 nucleotide 5´ overhanging ends. EnGen Lba Cas12a has Simian virus 40 (SV40) T antigen nuclear localization sequences (NLS) at both the N and C-termini of the protein. 

Required Materials:

  • EnGen Lba Cas12a (Cpf1) (NEB #M0653)

  • 10X NEBuffer 2.1 Reaction Buffer

  • Nuclease-free water

  • Proteinase K, Molecular Biology Grade (NEB #P8107)

  • Guide RNA containing the targeting sequence in the region of interest

  • 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
    • 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 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 Cas12a and gRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here.

  • Prepare 300 nM gRNA 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 EnGen Lba Cas12a (NEB #M0653T) for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X Buffer 2.1 (NEB #B7202) prior to the reaction assembly and used immediately. If the 1 μM dilution will be stored at -20°C, it should be diluted using the enzyme storage buffer: 500 mM NaCl, 20 mM sodium acetate, 0.1 mM EDTA, 0.1 mM TCEP and 50% glycerol (pH 6.0 @ 25°C).

Procedure:

  1. Assemble the reaction at room temperature in the following order*:

    COMPONENT  AMOUNT
    Nuclease-free water   20 µl
    NEBuffer 2.1 Reaction Buffer (10X)   3 µl
    300 nM gRNA
      3 µl (30 nM final) 
    1 µM EnGen Lba Cas12a (Cpf1)
      1 µl (~30 nM final) 
    Total Reaction Volume  27 µl


  2. Pre-incubate for 10 minutes at 25⁰C.

  3. Add 3 µl of 30 nM substrate DNA (30 µl final volume).

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

  5. Incubate at 37°C for 10 minutes.

  6. Add 1 µl of Proteinase K (NEB #P8107) to each sample, Mix thoroughly and pulse-spin in a microfuge. 

  7. Incubate at room temperature for 10 minutes.

  8. Proceed with analysis. 

References:

  1. Zetsche, B., et. al. (2015) Cel,l 163, 759-771.