Genome Editing
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A recombinant Cas9 enzyme that drastically reduces CRISPR off-target effects

To address concerns of off-target editing commonly observed in CRISPR-Cas9 applications, IDT has developed a high-fidelity, S. pyogenes Cas9 enzyme variant. The recombinant Alt-R® S.p. HiFi Cas9 Nuclease 3NLS offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events during CRISPR genome editing. This Cas9 variant also preserves the high level of editing efficiency at intended on-target sites expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites.

For best results, we recommend combining the Alt-R S.p. HiFi Cas9 Nuclease with optimized Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA, and delivering them in a ribonucleoprotein (RNP) format. Figure 1 demonstrates on-target vs off-target genome editing at 3 loci for wild-type Cas9 (dark blue) compared to 3 Cas9 variants, all delivered in an RNP format, in this case, by lipofection. (Note that as for Alt-R S.p. Cas9 Nuclease 3NLS, RNPs containing Alt-R S.p. HiFi Cas9 Nuclease are also effective when delivered by electroporation or microinjection.) All of the Cas9 variants showed strong discrimination between on-target and off-target sites, however the Alt-R S.p. HiFi Cas9 Nuclease 3NLS enzyme (orange) also consistently provided the highest on-target editing, comparable to that of wild-type Cas9.

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 (Alt-R Genome Editing Detection Kit;IDT) 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 = alternative base in PAM site).

Stanford University researcher compares high fidelity Cas9 enzymes

“We performed an unbiased evaluation of several versions of high fidelity Cas9 enzymes in primary human stem cells. We have been very impressed with the characteristics of this new IDT enzyme. Unlike other versions, this version consistently gives us high on-target editing activity while having low off-target activity. Because of the retained excellent on-target activity and improved specificity profile, we are excited to use this version in our future experiments focused on developing novel genome editing based therapies for severe diseases with unmet medical needs.”

– Matthew Porteus, MD, PhD
Division of Stem Cell Transplantation and Regenerative Medicine
Stanford University
Stanford, CA USA

The Alt-R S.p. HiFi Cas9 Nuclease 3NLS is purified from an E. coli strain expressing codon optimized Cas9. It contains 1 N-terminal nuclear localization sequence (NLS), 2 C-terminal NLSs, and a C-terminal 6-His tag, and is available in 100 and 500 µg aliquots (100 µg Cas9 nuclease = 610 pmol).

Learn more about Alt-R S.p. HiFi Cas9 Nuclease 3NLS.

Product focus—genome editing with Alt-R CRISPR Reagents

Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection, electroporation, or microinjection
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.

Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System recognizes an AT-rich PAM site, providing CRISPR target sites that are not available with the CRISPR-Cas9 System. In addition, Cpf1 nuclease produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation

Note that Cpf1 reagents are not interchangeable with Cas9 reagents, because of different requirements for PAM sequences and guide RNAs.

Learn more about the Alt-R CRISPR-Cpf1 System.

CRISPR support tools

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.

Related reading

Using CRISPR genome editing for gene knockout and homology-directed repair (HDR)—Webinar review: Watch our webinar recording for expert guidance on a complete CRISPR genome editing workflow, including available tools and protocols. Also, see what we have learned about homology-directed repair and a new option for repair templates.

Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)—Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation.

CRISPR genome editing: 5 considerations for target site selection—Learn how your genome editing experiments can be improved with 5 quick tips for target selection and with reagents from the Alt-R CRISPR-Cas9 System.

Review other DECODED Online newsletter articles on CRISPR in genome editing applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Ellen Prediger, PhD, is a scientific writer at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see

CRISPR-Cas9 Genome Editing

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