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CRISPR nucleases

Recombinant, high-purity endonucleases for genome editing experiments

Choose from Cas9 (Streptococcus pyogenes) and Cas12a (Cpf1; Acidaminococcus sp. BV3L6) proteins

  • Nuclear localization signals (NLSs) provide 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

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Which Cas9 enzyme to use

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

Alt-R S.p Cas9 nuclease

Alt-R S.p. Cas9 Nuclease V3 is a high purity, recombinant S. pyogenes Cas9. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The S. pyogenes Cas9 enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease V3 enzyme with optimized Alt-R CRISPR gRNA in equimolar amounts.

Alt-R S.p. HiFi Cas9 nuclease

Alt-R S.p. HiFi Cas9 Nuclease V3 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 V3 with the optimized Alt-R CRISPR-Cas9 gRNA (crRNA: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 either Alt-R S.p. Cas9 D10A Nickase V3 or Alt-R S.p. Cas9 H840A Nickase V3, with 2 gRNAs that target two neighboring Cas9 sites, one on either strand of the target region. This functionally increases the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the application note.

Alt-R S.p. dCas9 protein

Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA.

Alt-R A.s. Cas12a (Cpf1) nuclease

Alt-R A.s. Cas12a (Cpf1) Nuclease V3 enzyme is a high purity, recombinant Acidaminococcus sp. BV3L6 Cas12a. 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.

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

 Cas9 systemCas12a 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
  • gRNA options:
    1. crRNA and tracrRNA
    2. sgRNA
  • Cas9 endonuclease
  • crRNA
  • Cas12a endonuclease
Variants
  • Wild-type
  • HiFi
  • Nickases (H840A and D10A)
Wild-type
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 sequenceNGGTTTV
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

This comparison table is available for download (see page 2).

Cas9 Nuclease

Improved editing efficiency using Alt-R S.p. Cas9 Nuclease V3

Alt-R S.p. Cas9 Nuclease V3 is designed to maximize the efficiency of genome editing across a broad number of sites. Modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced cleavage, particularly at difficult targets (Figure 1).

Figure 1. Alt-R S.p. Cas9 Nuclease V3 maximizes genome editing efficiency even at challenging target sites. Ribonucleoprotein (RNP) complexes were formed with 1 of the 2 wild-type Cas9 proteins—Alt-R S.p. Cas9 Nuclease 3NLS (light blue) or Alt-R S.p. Cas9 Nuclease V3 (dark blue), combined with an Alt-R crRNA:tracrRNA complex targeting one of 11 loci on the human HPRT gene. RNP complexes (4 µM) were delivered into HEK-293 cells by nucleofection. Total editing at the on-target loci was calculated by NGS.

Increased specificity using Alt-R S.p. HiFi Cas9 Nuclease V3

As with the wild-type Alt-R Cas9 Nuclease V3, modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced on-target cleavage by Alt-R S.p. HiFi Cas9 Nuclease V3. However, Alt-R HiFi Cas9 Nuclease V3 also provides superior cutting specificity (minimized off-target editing; Figure 2).

Figure 2. Alt-R S.p. HiFi Cas9 Nuclease V3 facilitates near-WT on-target editing potency and significantly reduces off-target site editing. RNP complexes were formed with either Alt-R S.p. Cas9 Nuclease V3 or Alt-R S.p. HiFi Cas9 Nuclease V3, combined with an Alt-R crRNA:tracrRNA complex targeting the EMX1 gene. RNP complexes (4 µM) were delivered into HEK-293 cells via nucleofection. INDEL formation at the on-target locus as well as 9 known off-target sites were measured by NGS (indicated on the y axis in log scale).

Potent editing with the Alt-R S.p. Cas9 nucleases

The Alt-R CRISPR-Cas9 System includes potent Alt-R S.p. Cas9 nucleases. When Alt-R S.p. Cas9 Nuclease 3NLS was combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperformed other editing approaches (Figure 3). You can expect even better editing efficiency with Alt-R S.p. Cas9 Nuclease V3 (see 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 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.

 

Cas12a Nuclease

Improved Alt-R Cas12a V3 enzyme dramatically increases overall editing efficiency

To enhance performance, we introduced multiple modifications to the Cas12a protein that resulted in remarkable improvement in overall editing efficiency. These changes resulted in remarkable improvement in overall editing efficiency of the Cas12a protein. Figure 4 demonstrates the difference in total editing efficiency between the original Alt-R A.s. Cpf1 Nuclease 2NLS and modified Alt-R A.s. Cas12a Nuclease V3 on 96 sites distributed over 24 genes.

Figure 4. Optimal genome editing efficiency with a novel Cas12a (formerly known as Cpf1) endonuclease. (A) 96 sites distributed over 24 genes were targeted by ribonucleoprotein (RNP) complexes formed with Alt-R CRISPR-Cpf1 (Cas12a) crRNA:tracrRNA together with either Alt-R A.s. Cas12a Nuclease V3 (dark blue) or Alt-R A.s. Cpf1 Nuclease 2NLS (light blue). RNPs were delivered at 5 µM concentration together with 3 µM Alt-R Cpf1 (Cas12a) Electroporation Enhancer using the Amaxa Nucleofector (Lonza). Total editing efficiencies were determined 48 hours after electroporation by NGS-based targeted sequencing. Target sites are ranked based on increasing editing efficiency. (B) Aggregated editing efficiencies from using Alt-R A.s. Cas12a Nuclease V3 (dark blue) are compared to that of Alt-R A.s. Cpf1 Nuclease 2NLS (light blue) at the 96 loci shown in Figure 4A.

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

We have found that some of the Cas12a PAM sequences are not active sites for genome editing (Figure 5). We recommend that you test 3 or more PAM sites in your region of interest and include the Alt-R Cas12a (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.

Figure 5. 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, Amaxa® Nucleofector® system (available at www.idtdna.com/CRISPR-Cpf1). 12 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 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.

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 even higher performance. The latest versions (V3) can be directly substituted into the protocols in place of the prior Alt-R enzymes.

Alt-R CRISPR-Cas9 System

Alt-R CRISPR-Cpf1 System

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