Product Class: Other

EnGen® Lba Cas12a (Cpf1)
neb31 recombinant 37 65 Heat

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

Product Introduction

EnGen Lba Cas12a (Cpf1) is a programmable DNA endonuclease guided by a single guide RNA (gRNA). Targeting requires a gRNA complementary to the target site as well as a 5' TTTV protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Cleavage by EnGen Lba Cas12a (Cpf1) occurs ~17 bases 3' of the PAM and leaves 5' overhanging ends.

  • T-rich TTTV PAM sequence opens up additional genomic regions for targeting
  • Shorter, 40-44 base guide RNA
  • Two nuclear localization signals for improved transport to the nucleus
  • 5’ overhanging termini on cleavage products
  • Active from 16 to 48°C
  • Maintains activity at lower temperatures than the Acidaminococcus orthologs, permitting editing in ectothermic organisms such as zebra fish and xenopus
  • High concentration liquid format can be used for microinjection, electroporation and lipofection.
Catalog # Size Concentration
M0653S 70.0 pmol 1 µM
M0653T 2000.0 pmol 100 µM

Product Information

Description

EnGen® Lba Cas12a 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’ TTTV 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′ 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.

 

Schematic representation of Lba Cas12a nuclease sequence recognition and DNA cleavage

Cas12a


Expanded temperature range of Lba Cas12a

Cas12a Temperature

Purified Lba or Asp Cas12a proteins were used to form RNP complexes with their canonical guide RNAs. The DNA target is a single a PCR product 5´-end labeled with FAM. Lba or Asp Cas12a RNPs were incubated with target DNA at the temperatures indicated at a ratio of 10:1 RNP to DNA for 10 minutes. Cleavage products were resolved by capillary electrophoresis and quantitated by integration of signal corresponding to intact and cleaved fragments. Data are shown as the mean +/- SD of duplicate experiments.

Product Source

EnGen Lba Cas12a from Lachnospiraceae bacterium ND2006 is expressed as a N-terminal 6XHis-tagged fusion in E. coli.
This product is related to the following categories:
Genome Editing Products,
Programmable Nucleases Products,
This product can be used in the following applications:
Genome Editing Applications

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Reaction Conditions

1X NEBuffer™ r2.1
Incubate at 37°C

1X NEBuffer™ r2.1
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Storage Buffer

500 mM NaCl
20 mM sodium acetate
0.1 mM EDTA
0.1 mM TCEP
50% Glycerol
pH 6 @ 25°C

Heat Inactivation

65°C for 10 minutes

Product Notes

  1. 100 µM is equal to 15.1 mg/ml EnGen Lba Cas12a.

References

  1. Zetsche, B., et. al. (2015). Cell. 163, 759-771.
  2. Moreno-Mateos, M.A., et al. (2017). Nat. Commun.. 8, 2024. PubMedID: 29222508

Protocols, Manuals & Usage

Protocols

  1. Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP) into adherent cells using the Neon® Electroporation
  2. Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP) into adherent cells using the Lonza 4D-Nucleofector®
  3. In vitro digestion of DNA with EnGen® Lba Cas12a (Cpf1) (M0653)

FAQs & Troubleshooting

FAQs

  1. What are the differences between EnGen® Lba Cas12a (Cpf1) (NEB #M0653) and EnGen Spy Cas9 NLS (NEB #M0646)?
  2. Which nuclear localization signal is fused to EnGen® Lba Cas12a (Cpf1)?
  3. Why am I observing limited or no digestion with EnGen® Lba Cas12a (Cpf1)?
  4. Why does digestion/editing efficiency differ between two different guide RNAs?
  5. Can mutations generated with EnGen Lba Cas12a (Cpf1) be detected using T7 Endonuclease I (NEB #M0302) or the EnGen Mutation Detection Kit (NEB #E3321S)?
  6. Can gRNA for use with EnGen Lba Cas12a (Cpf1) be generated using the EnGen sgRNA Synthesis Kit, S. pyogenes (NEB #E3322)?
  7. How do I design a guide RNA for use with EnGen Lba Cas12a?
  8. How do I dilute the enzyme to 1 μM for in vitro reactions?
  9. Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers?
  10. What is in EnGen Lba Cas12a Diluent (NEB #B0653A)?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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