In vitro digestion of DNA with EnGen® Lba Cas12a (Cpf1) (NEB #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
o  Agarose gel electrophoresis apparatus
 DNA Loading Dye (e.g., Gel Loading Dye, Purple (6X) (NEB #B7024S)
o 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 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.

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
*The substrate DNA and gRNA, and nuclease-free water are not included.
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.