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.


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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.

Cas9 nuclease comparison table

Comparison of Alt-R Cas9 Nucleases and Nickases. Click here to download PDF version.

Alt-R S.p Cas9 Nuclease 3NLS

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.

Alt-R S.p. HiFi Cas9 Nuclease 3NLS

Alt-R S.p. HiFi Cas9 Nuclease offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease with the optimized Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA is highly recommended.

Alt-R S.p. Cas9 Nickases

Cas9 nickases allow specific cutting of only one strand at the DNA target site. Cuts to both strands of DNA are accomplished by using both the Alt-R® S.p. Cas9 D10A and the Alt-R S.p. Cas9 H840A Nickase 3NLS, and targeting two neighboring Cas9 sites, functionally increasing the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the Application Note.

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.

 

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
Variants• Wild-type
• HiFi
• Nickases (H840A and D10A)
Wild-type
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 recommended)
• Microinjection
• Electroporation (Alt-R enhancer required)
• Microinjection

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


Increased specificity using Alt-R® S.p. HiFi Cas9 Nuclease 3NLS

To address concerns of off-target editing commonly observed in CRISPR-Cas9 applications, IDT has developed a high-fidelity, S. pyogenes Cas9 nuclease. This novel Cas9 variant drastically reduces activity at off-target sites, while maintaining the high potency of wild-type Cas9 at the intended on-target site (Figure 1 and 2).

Cas9 wt vs HiFi

Figure 1. Alt-R S.p. HiFi Cas9 Nuclease 3NLS provides consistently high on-target performance, while reducing off-target editing, when delivered as a ribonucleoprotein (RNP). RNP complexes (1 µM) were formed with wild-type Cas9 protein (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) and 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88), combined with an Alt-R crRNA:tracrRNA gRNA complex targeting the EMX1, HEKSite4, or VEGFA3 loci. Complexes (10 nM) were delivered into HEK-293 cells by reverse transfection using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher), and DNA was extracted after 48 hr. Editing was measured by PCR amplification of the indicated on- and off-target sites, followed by cleavage with T7EI and analysis using the Fragment Analyzer™ system (Advanced Analytical). Error bars represent the standard errors of the means. The sequence of the on- and off-target sites for each crRNA are indicated at the bottom (red = mismatch in protospacer; blue = mismatch in PAM site).

Alt-R® HiFi Cas9 similar editing efficiency to wild-type Cas9 v2

Figure 2. Alt-R HiFi Cas9 maintains similar on-target editing efficiency to wild-type Cas9. Alt-R crRNAs targeting 12 sites within the human HPRT gene were annealed with Alt-R tracrRNA, then complexed to form ribonucleoprotein (RNP) editing complexes with wild-type Cas9 (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) or one of 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88). The RNP complexes were reverse transfected using Lipofectamine® RNAiMAX™ reagent (Thermo Fisher) into HEK-293 cells at a concentration of 10 nM. After 48 hr, genomic DNA was isolated, and editing at the intended on-target site was measured by a T7 endonuclease I assay (Alt-R Genome Editing Detection Kit). The Alt-R S.p. HiFi Cas9 maintains higher levels of editing across different target sites when compared to previously published, enhanced specificity Cas9 mutants.

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 3). 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 3. 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.

Improved homology-directed gene repair using the Alt-R CRISPR-Cas9 System RNP

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 (Figure 4). 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 4. 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.