CRISPR nucleases

Recombinant, high-purity endonucleases for genome editing experiments

Choose from Cas9 (Streptococcus pyogenes) and Cpf1 (Acidaminococcus sp. BV3L6).

  • Contain nuclear localization signals (NLSs) for increased genome editing efficiency
  • Use as part of a ribonucleoprotein (RNP) complex for optimal performance
  • Codon-optimized for expression in E. coli resulting in high-purity enzymes

This page allows quick ordering of Cas9 or Cpf1 nucleases. For more information about additional reagents for CRISPR genome editing, visit the product pages for the Alt-R® CRISPR-Cas9 System or Alt-R CRISPR-Cpf1 System.


This page allows quick ordering of Cas9 or Cpf1 nucleases.

The end of this section contains a comparison of CRISPR-Cas9 and -Cpf1 systems. For more information about additional reagents for CRISPR genome editing, visit the product pages for the Alt-R® CRISPR-Cas9 System or Alt-R CRISPR-Cpf1 System.


Alt-R® S.p Cas9 Nuclease 3NLS

The Alt-R S.p. Cas9 Nuclease 3NLS enzyme is a high purity, recombinant S. pyogenes Cas9. The enzyme includes 1 N-terminal nuclear localization sequence (NLS) and 2 C-terminal NLSs, as well as a C-terminal 6-His tag. The S. pyogenes Cas9 enzyme must be combined with a crRNA and tracrRNA in order to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease 3NLS enzyme with the optimized Alt-R CRISPR crRNA and tracrRNA in equimolar amounts.

Product specifications:

Alt-R® S.p Cas9 Nuclease 3NLS 

  • Amount provided: 100 µg or 500 µg
  • Molecular weight: 163,700 g/mol
  • Concentration: 10 µg/µL in 50% glycerol, [61 µM]
  • Endotoxin tested: <2 EU/mg
  • Shipping conditions: dry ice
  • Storage conditions: –20°C

Alt-R® A.s. Cpf1 Nuclease 2NLS

Alt-R A.s. Cpf1 Nuclease 2NLS enzyme is a high purity, recombinant Acidaminococcus sp. BV3L6 Cpf1. The enzyme includes 1 N-terminal nuclear localization sequence (NLS) and 1 C-terminal NLSs, as well as 3 N-terminal FLAG tags and a C-terminal 6-His tag. The Cpf1 enzyme must be combined with a crRNA to produce a functional, target-specific editing complex. For the best editing, combine Alt-R A.s. Cpf1 Nuclease 2NLS enzyme with optimized Alt-R CRISPR-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. Cpf1 nuclease. We recommend using positive control crRNAs to establish that your cells can be edited by Cpf1. In addition, we suggest testing 3 or more crRNAs per target gene.

 

Product specifications:

Alt-R® A.s. Cpf1 Nuclease 2NLS 

  • Amount provided: 100 µg or 500 µg
  • Molecular weight: 157,900 g/mol
  • Concentration: 10 µg/µL in 50% glycerol, [63 µM]
  • Endotoxin tested: <2 EU/mg
  • Shipping conditions: dry ice
  • Storage conditions: –20°C

Comparison of CRISPR genome editing using Cas9 vs. Cpf1


  Cas9 system Cpf1 system
ApplicationsGeneral genome editing• For species with AT-rich genomes
• For regions with limiting design space for use of the CRISPR-Cas9 system
Ribonucleoprotein components • crRNA
• tracrRNA
• Cas9 endonuclease
• crRNA
• Cpf1 endonuclease
crRNA• Native: 42 nt
• Alt-R: 35–36 nt (36 nt recommended)
• Native: 42–44 nt
• Alt-R: 40–44 nt (41 nt recommended)
tracrRNA• Native: 89 nt
• Alt-R: 67 nt
 (not applicable)
CRISPR enzyme• Class 2, Cas type II
• M.W.*: 163,700 g/mol
• Endonuclease domains: RuvC-like and HNH 
• Class 2, Cas type V
• M.W.*: 157,900 g/mol
• Endonuclease domain: RuvC-like only
Double-stranded DNA cleavage • Blunt ended cut 3 bases upstream of the protospacer sequence
• 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
Current recommendations for Alt-R® RNP delivery • Lipid-mediated transfection
• Electroporation ± Alt-R® enhancer 
• Microinjection
• Electroporation with Alt-R® enhancer
• Microinjection

* Molecular weight of Alt-R® nuclease
N = any base; V = A, C, or G


Potent editing with the Alt-R® S.p. Cas9 Nuclease 3NLS

The Alt-R CRISPR-Cas9 System includes the potent Alt-R S.p. Cas9 Nuclease 3NLS. When Alt-R S.p. Cas9 Nuclease 3NLS is combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperforms other editing approaches (Figure 2). RNP transfections also provide optimal control of dose of editing complexes, and the non-renewable Cas9 RNP is cleared after a short duration by endogenous mechanisms, limiting off-target editing.

Figure 1. Lipofection of Alt-R® CRISPR-Cas9 System components as a ribonucleoprotein outperforms other transient CRISPR-Cas9 approaches. Alt-R CRISPR HPRT Control crRNA complexes for human, mouse, or rat were complexed with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as part of a Cas9 RNP (containing Alt-R S.p. Cas9 Nuclease 3NLS, pre-complexed with the crRNA and tracrRNA) into human (HEK-293), mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression approaches, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.

Case study

View related data shared by Dr Eric Kmiec (Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health System, Wilmington, DE, USA):


The electroporation enhancer is required for efficient genome editing with the CRISPR-Cpf1 system

We have found that some of the Cpf1 PAM sequences are not active sites for genome editing. We recommend that you test 3 or more PAM sites in your region of interest and include the Alt-R® Cpf1 Electroporation Enhancer for efficient genome editing. The enhancer is a non-targeting carrier DNA that shows no integration into the target site based on next generation sequencing experiments.

Alt-R CRISPR-Cpf1 Electroporation Enhancer is required for RNP electroporation

Figure 2. Alt-R® 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-Cpf1 crRNA) as instructed in the Alt-R CRISPR-Cas9 User Guide—RNP electroporation, Amaxa® Nucleofector® system (available at www.idtdna.com/CRISPR-Cpf1). 12 Cpf1 PAM sites in the HPRT gene were targeted by Alt-R CRISPR-Cpf1 crRNAs. The electroporation reactions contained either no (dark blue) or 3 µM (light blue) Alt-R Cpf1 Electroporation Enhancer. Editing efficiency 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 (Cpf1 PAM sequence is TTTV); x-axis: numbers specify gene locations; S = sense strand; AS = antisense strand.