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Cas12a proteins

Harness the power of the Alt-R CRISPR-Cas12a enzymes to target genomic regions beyond the reach of Cas9. Explore our range of Cas12a (Cpf1) nucleases, including the proprietary Alt-R Cas12a Ultra, designed for enhanced editing precision, especially in AT-rich genomes. Achieve effective editing with staggered 5' overhangs and streamlined crRNA complexing—no tracrRNA required. Available for L.b. and A.s. Cas12a crRNAs.
DNA Strand

Ordering

  • Access additional genomic sites not available with Cas9, ideal for editing AT-rich genomes.
  • Proprietary Alt-R Cas12a Ultra nucleases offering enhanced editing efficiency and precision, with activity at low temperatures—perfect for editing in ectothermic organisms.
  • Reduce off-target editing with Alt-R Cas12a Ultra nucleases, making them ideal for precise and sensitive genome editing applications

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Custom CRISPR solutions

Don’t see what you’re looking for? We are continually expanding our CRISPR product line, and we may have what you need. If you are interested in custom libraries, other CRISPR enzymes, formulations, or other CRISPR tools, contact our CRISPR experts today to discuss customized solutions for your research: CRISPR@idtdna.com.

Product Details

Cas12a (Cpf1) proteins

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA endonuclease that is an alternative to the commonly used Streptococcus pyogenes Cas9 (S.p. Cas9) enzyme. Unlike S.p. Cas9, which recognizes NGG PAM sequences, Cas12a recognizes TTTV (V = A/G/C) PAM sites, thereby permitting genome editing in organisms with AT-rich genomes. A.s. Cas12a is an attractive option for genome editing applications due to its AT-rich PAM sequence, its highly specific DNA recognition and cleavage mechanism, and its native reliance on a single, short guide RNA.

Alt-R A.s. Cas12a (Cpf1) V3 nuclease
Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme is a high purity, recombinant Acidaminococcus sp. BV3L6 Cas12a. It is useful for targeting AT-rich regions when the Cas9-specific PAM sequence (NGG) is not available. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The Cas12a enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme with optimized Alt-R CRISPR-Cas12a (Cpf1) crRNA in equimolar amounts.

Attention: Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cas12a nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cas12a. In addition, we suggest testing 3 or more crRNAs per target gene.

Alt-R A.s. or L.b. Cas12a (Cpf1) Ultra Nucleases
The Alt-R Cas12a (Cpf1) Ultra Nucleases are also useful for targeting AT-rich regions without available Cas9-specific PAM sequences.  However, they have much higher on-target potency than wild-type A.s. Cas12a (Cpf1). The Alt-R Cas12a (Cpf1) Ultra also can recognize many TTTT PAM sites in addition to TTTV motifs, increasing target range for genome editing studies. Furthermore, the new Alt-R Cas12a (Cpf1) Ultra nucleases are active at room temperature, making them flexible tools for applications requiring delivery at lower temperatures.

Comparison of CRISPR genome editing using Cas9 vs. Cas12a (Cpf1)

Cas9 system Cas12a system
Applications
  • General genome editing
  • For species with AT-rich genomes
  • For regions with limiting design space for use of the CRISPR-Cas9 system
Ribonucleoprotein components
  • gRNA options:
    1. crRNA and tracrRNA
    2. sgRNA
  • Cas9 endonuclease
  • crRNA
  • Cas12a endonuclease
Variants
  • Wild-type
  • HiFi
  • Nickases (H840A and D10A)
  • Cas9-GFP (or RFP)
  • Wild-type
  • Ultra (improved performance)
Cas9 crRNA:tracrRNA (option 1) crRNA
  • Native: 42 nt
  • Alt-R: 35–36 nt (36 nt recommended)
tracrRNA
  • Native: 89 nt
  • Alt-R: 67 nt
Cas9 sgRNA (option 2)
  • Alt-R: 99–100 nt (100 nt recommended)
Cas12a crRNA
  • Native: 42–44 nt
  • Alt-R: 40–44 nt (41 nt recommended)
CRISPR enzyme
  • Class 2, Cas type II
  • M.W.*: 162,200 g/mol
  • Endonuclease domains: RuvC-like and HNH
  • Class 2, Cas type V
  • M.W.*: 156,400 g/mol
  • Endonuclease domain: RuvC-like only
DNA cleavage
  • Wild-type and HiFi: Blunt-ended cut 3 bases upstream of the protospacer sequence
  • D10A nickase with paired gRNAs: 5′ overhang
  • H840A nickase with paired gRNAs: 3′ overhang
  • PAM site often destroyed during genome editing
  • 5′ overhanging cut on the 5′ side of the protospacer sequence
  • PAM site may be preserved after genome editing
PAM sequence†
  • NGG
  • TTTV for Cas12a V3
  • TTTN for Cas12a Ultra
Current recommendations for Alt-R RNP delivery
  • Lipid-mediated transfection
  • Electroporation (Alt-R enhancer recommended)
  • Microinjection
  • Electroporation (Alt-R enhancer recommended)
  • Microinjection
* Molecular weight of Alt-R nuclease
† N = any base; V = A, C, or G

Product Data

Newly developed Alt-R Cas12a (Cpf1) Ultra enzyme increases overall editing efficiency

To enhance activity, we introduced multiple modifications to the Cas12a protein that support notable improvement in overall editing efficiency. The new Alt-R Cas12a (Cpf1) Ultra nuclease has higher on-target potency than the wild-type A.s. Cas12a (Cpf1). The new Alt-R Cas12a (Cpf1) Ultra also can recognize many TTTT PAM sites in addition to TTTV motifs, increasing target range for genome editing studies (Figure 1). Furthermore, the new Alt-R Cas12a (Cpf1) Ultra nuclease is active at room temperature, making it a flexible tool for applications requiring delivery at lower temperatures.

Figure 1. New A.s. Cas12a (Cpf1) Ultra exhibits increased genomic editing efficiency in Jurkat and HEK-293 cells. Ribonucleoprotein (RNP) complexes were formed with wild type (WT) or Alt-R A.s. Cas12a (Cpf1) Ultra (Ultra), combined with crRNAs synthesized for 120 genomic loci to be delivered in Jurkat cells and 96 genomic loci to be delivered in HEK-293 cells. RNP complexes (4 μM) were delivered into Jurkat and HEK-293 cells via a Nucleofector™ system (Lonza) in the presence of Alt-R Cas12a (Cpf1) Electroporation Enhancer. Genome editing efficiencies were determined by target amplification followed by next generation sequencing on an Illumina instrument. The Cas12a-associated PAM sequences are indicated below the graph. n = 426, with 213 data points for WT and 213 data points for Cas12a Ultra. Each dot represents a single sample.

The electroporation enhancer is recommended for efficient genome editing with the CRISPR-Cas12a (Cpf1) system

The Alt-R Cas12a (Cpf1) Electroporation Enhancer is a Cas12a-specific carrier DNA that is optimized to work with the Nucleofector™ device (Lonza) and the Neon™ Transfection System (Thermo Fisher) for increased transfection efficiency and therefore, increased genome editing efficiency (Figure 2). The electroporation enhancer is non-targeting and shows no integration into the target site based on next-generation sequencing experiments.

Figure 2. Alt-R Cas12a (Cpf1) Electroporation Enhancer is required for efficient CRISPR editing in ribonucleoprotein (RNP) electroporation experiments. HEK-293 cells were electroporated with 5 µM RNP (Alt-R A.s. Cpf1 Nuclease 2 NLS complexed with Alt-R CRISPR-Cas12a (Cpf1) crRNA) as instructed in the Alt-R CRISPR-Cas12a (Cpf1) User Guide—RNP electroporation, Nucleofector™ system (available at www.idtdna.com/CRISPR-Cpf1). Twelve Cas12a PAM sites in the HPRT gene were targeted by Alt-R CRISPR-Cas12a (Cpf1) crRNAs. The electroporation reactions contained either no (dark blue) or 3 µM (light blue) Alt-R Cas12a (Cpf1) Electroporation Enhancer. Editing efficiency (n =3) was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required for T7EI endonuclease assays. PAM = protospacer adjacent motif (Cas12a PAM sequence is TTTV); x-axis: numbers specify gene locations; S = sense strand; AS = antisense strand.

Resources

User guides and protocols

Improved enzymes: All Alt-R enzymes [Cas9 nuclease, HiFi Cas9 nuclease, Cas9 nickases, and Cas12a (Cpf1) nuclease] have recently been further optimized to deliver accurate results in your research. The latest versions (Alt-R S.p. Cas9 Nuclease V3 and A.s. Cas12a (Cpf1) Ultra) can be directly substituted into the protocols in place of the prior Alt-R enzymes.

Alt-R CRISPR-Cas12a System - Delivery of ribonucleoprotein complexes into HEK-293 cells using the Amaxa Nucleofector System Protocol
(845 KB)
Alt-R CRISPR-Cas12a Cpf1 System: Delivery of ribonucleoprotein complexes into Jurkat T cells using the Neon Transfection System Protocol
(672 KB)

Application notes and case studies

Certificates of analysis (COAs)

Find COAs by batch or lot number

Frequently Asked Questions

All Cas12a variants are provided as a 10 mg/mL solution and, therefore, resuspension is not required.
View FAQ

For the Alt-R™ CRISPR-Cas12a crRNA, the input sequence can vary between 20 and 24 nucleotides. However, based on internal studies, we recommend using a 21-nucleotide sequence as input into the CRISPR-Cas12a crRNA ordering tool.

The ordering tool will automatically convert the DNA sequence to RNA during the ordering process. The 20- or 21-base constant region (loop domain) for the selected Cas12a nuclease (A.s. or L.b. Cas12a) and modifications that confer intracellular nuclease resistance will automatically be added. The final Alt-R CRISPR-Cas12a crRNA will be 40–45 bases.

View FAQ

A.s. Cas12a is from Acidaminococcus sp. BV3L6, and L.b. Cas12a is from Lachnospiraceae bacterium. Both utilize the same TTTV PAM site and crRNA design considerations.

Both Cas12a nucleases result in robust editing at target sites at 37°C, with L.b. Cas12a also resulting in high editing efficiencies in plant genomes and at lower temperatures, such as 23°C.

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Our Alt-R Cas12a Nucleases should be stored at –20°C.

Under optimal storage conditions, these proteins maintain functionality for at least 1-2 years. See our stability study for more details.

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We have successfully used electroporation in internal experiments to deliver Cas12a RNP complexes to cells in culture. In many cell lines, Alt-R™ Cas12a Electroporation Enhancer, a non-targeting carrier DNA, is also required for efficient delivery of the RNP complex. IDT protocols using Cas12a and electroporation are included here, and these Cas12a DECODED articles include functional data and details:

View FAQ

Alt-R Cas12a (Cpf1) Electroporation Enhancer improves electroporation efficiency and is a single-stranded DNA oligonucleotide that was computationally designed to be non-homologous to human, mouse, or rat genomes.

The use of this enhancer is important for efficient electroporation of ribonucleoproteins (RNPs), which, in turn, is important for increased rates of editing.

View FAQ

We recommend delivering a ribonucleoprotein (RNP), consisting of Cas12a (Cpf1) nuclease pre-complexed to crRNA.

Non-targeting carrier DNA (i.e., Alt-R™ Cpf1 Electroporation Enhancer) can also be included in the electroporation for efficient editing.

View FAQ

Stability studies for the Alt-R Cas12a RNP complex have found that the Cas12a RNP complex is stable for up to 2 months at 4°C and for up to 1 year at –20°C.


Additional details are included in our CRISPR reagents stability DECODED article.

View FAQ

We have observed that dose response curves vary for different guide sequences, depending on potency of the guide RNA. We recommend titrating the amount of ribonucleoprotein (RNP) and keeping the amount of electroporation enhancer fixed.

For the Nucleofector™ System (Lonza), we recommend 3 µM of Alt-R Cas12a Electroporation Enhancer, and for the Neon™ System (Thermo Fisher Scientific), we recommend 1.8 µM of Alt-R Cas12a Electroporation Enhancer. Toxicity may be observed at high concentrations of enhancer.

View FAQ

The PAM sequence for the Cas12a (Cpf1) system is TTTV [1], where "V" is a A, C, or G. The Cas12a PAM sequence can be advantageous when working with T-rich target sequences. In contrast, the Cas9 PAM sequence is NGG.

Reference

  1. Zetsche B, Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759-771.
View FAQ

The Alt-R™ CRISPR-Cas12a (Cpf1) System includes a target-specific CRISPR-Cas12a crRNA, Cas12a (Cpf1) endonucleases (Alt-R A.s.‑ Cas12a (Cpf1) V3, Alt-R A.s. Cas12a (Cpf1) Ultra, or Alt-R L.b. Cas12a (Cpf1) Ultra), and carrier DNA (Alt-R Cpf1 Electroporation Enhancer).

You will need to supply your own electroporation reagents to effectively deliver CRISPR reagents into your cell lines.

We recommend delivery of these reagents as a ribonucleoprotein  (RNP). However, Alt-R A.s. Cas12a crRNA also works well in cells that stably express Acidaminococcus sp. BV3L6 Cpf1 nuclease.

We also highly recommend the use of control crRNAs. Control sequences for the CRISPR-Cas12a (Cpf1) system are provided online and in the user guides accessible here.

View FAQ
Enter the 20–24 base DNA sequence downstream (3’ end) of the target Cas12a (Cpf1) PAM site (TTTV*), and then you are ready to continue. We typically recommend using a 21-nucleotide guide sequence. The ordering tool will automatically convert the DNA sequence to RNA during the ordering process. If you are pasting your Cas12a (Cpf1) target site from an online design tool, make sure to verify the correct strand orientation before pasting the target sequence.
how-to-order-alt-r-cpf1-crrnas
* V = A, C, or G
View FAQ
Our internal research data, utilizing next generation sequencing (NGS), showed a very low rate of integration of the enhancer into the target site (fewer than 1 in 10,000 reads, which is much lower than the insertion rate of random pieces of cellular DNA).
View FAQ
Check that you have included the Alt-R™ Cpf1 Electroporation Enhancer (carrier DNA) in the electroporation and that you are using one of our positive control crRNAs to monitor delivery efficiency. Also check that the target site in your cells does not show any polymorphism that could affect the potency of the crRNA. Target additional PAM sites in your gene of interest to identify a site that provides optimal editing efficiency.
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  • Table 1 shows available customizations for library design, formulations, and shipping formats.

Product Specifications

Features Options
Design Predesigned, custom, user-provided
CRISPR systems  Cas9, Cas12a, Cas13, prime editing, and other alternative systems
 Guaranteed Yield 0.5 nmol, 2 nmol, 5 nmol, and custom normalized deliverables
 Cas9 gRNA formats  2-part cRNA:tracrRNA complex and sgRNA
 Custom lengths supported  30-150 nt
 Chemical modifications  2'-O-methyl RNA, PS linkages, end-blocking Alt-R modifications
 Plate types  96- & 384-well PCR, Deep-well, V-bottom, ECHO, custom options available
 Formulation options  Multi-guide per well; pooled by gene Arrayed (single gRNA/well) Custom formulations upon request
 QC  Individual ESI/MS
Supporting reagents & functional analysis pipeline (optional)  WT Cas9, HiFi Cas9, Cas12a, and Cas12a Ultra Glycerol-free options available in tubes or plates (ideal for robotics) Electroporation Enhancers rhAmpSeq™ CRISPR Analysis System (NGS-based on-/off-target editing analysis)
  • Don’t see what you’re looking for? We are continually expanding our CRISPR library products and we may have what you need. If you are interested in other chemically modified gRNAs (such as CRISPR on/off systems) targeting any sequence from any species, email our CRISPR experts today to discuss customized solutions for your research at CRISPR@idtdna.com.
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