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Improve your genome editing with the Alt-R® S.p. Cas9 Nuclease 3NLS and modified crRNAs

CRISPR-Cas9 genome editing is changing the landscape of genomic research. The CRISPR-Cas9 mechanism relies on the Cas9 endonuclease to generate double-stranded breaks at specified sites in double-stranded DNA (dsDNA). The native Streptococcus pyogenes Cas9 protein that is most commonly used requires a 42 nt CRISPR RNA (crRNA) and an 89 nt transactivating CRISPR RNA (tracrRNA) to guide and activate the nuclease for target specific cleavage of dsDNA. The cleaved DNA is then repaired by one of two available cellular mechanisms: the error-prone non-homologous end-joining that is used to introduce frameshift mutations into protein coding sequences, leading to gene disruptions; and homology-directed recombination that is use to introduce foreign DNA into the genome.

Through extensive testing, scientists at IDT have developed the Alt-R® CRISPR-Cas9 System. The system combines a high quality S. pyogenes Cas9 protein with experimentally optimized, 36 nt crRNA and 67 nt tracrRNA oligonucleotides that outperform traditional CRISPR genome editing methods.

The Alt-R® S.p. Cas9 Nuclease 3NLS further improves genome editing

For optimal on-target editing efficiency, IDT offers the Alt-R S.p. Cas9 Nuclease 3NLS. The addition of this Cas9 protein gives researchers a powerful new tool that can be combined with Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP). The RNP is active upon delivery and is compatible with lipofection with commercially available reagents (see User Guide) or electroporation methods, and outperforms methods that rely on transfection of DNA expression constructs or Cas9 mRNA (Figure 1).

Alt-RTM CRISPR-Cas9 System ribonucleoprotein outperforms other transient CRISPR-Cas9 editing methods

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

In addition to improved editing performance, using RNP complexes, instead of DNA expression constructs or Cas9 mRNA, mitigates some of the problems associated with other methods [1,2]. For example, RNPs eliminate genomic incorporation issues associated with introducing double-stranded DNA expression constructs, and greatly reduces or eliminates toxicity observed when delivering long, in vitro transcribed RNAs. Also, because RNPs are non-renewable, this method allows for precise control over the dose of editing complexes, for more consistent editing results.

Updated Alt-R® CRISPR crRNAs with increased nuclease resistance

Alt-R CRISPR crRNAs have also been updated for improved performance through the addition of proprietary chemical modifications. These modifications provide further protection from degradation by cellular RNases, which improves on‑target editing performance in many applications. The modifications are included automatically in the final Alt-R CRISPR crRNA oligonucleotide sequence and do not require any changes to the ordering process.

The addition of the Alt-R S.p. Cas9 Nuclease 3NLS and the modified Alt-R CRISPR crRNA build on the already market leading Alt-R CRISPR-Cas9 system. The changes further improve performance in many applications and give researchers more options for implementing high performance genome editing without affecting current protocols. For more information, visit www.idtdna.com/CRISPR.


  1. Zuris JA, Thompson DB, et al. (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotech, 33(1):73–80.

  2. Ramakrishna S, Kwaku Dad AB, et al. (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res, 24(6):1020–1027.

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 or electroporation
  • 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 allows for new CRISPR target sites that are not available with the CRISPR-Cas9 System, and 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

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.

Additional resources

CRISPR guide RNA format affects genome editing outcomes—Learn how use of different formats for the guide RNAs associated with CRISPR-Cas9 genome editing can lead to different editing outcomes. The optimized, short RNA oligos that make up the crRNA and tracrRNA components of the Alt-R® CRISPR-Cas9 System outperform other CRISPR guide RNA formats. Unlike DNA expression constructs, short RNA oligos are unable to incorporate into the target genome, resulting in cleaner editing.

6 pieces of data that will change how you set up your CRISPR-Cas9 experiments—Discover how you can improve the efficiency of CRISPR-Cas9 genome editing. IDT scientists evaluated several factors that influence how we design and perform genome editing experiments. Review the data and results for 5 important factors that were addressed. These experimental findings resulted in a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.

Getting started with Alt-R® CRISPR-Cas9 genome editing—Webinar: Watch a recording of our webinar to learn about the components of the Alt-R CRISPR-Cas9 System, get information on designing Alt-R CRISPR crRNA oligos, and review the genome editing protocol from the user guide.

Author: Hans Packer, PhD, is a scientific writer at IDT.

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