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A novel, high-fidelity Cas9 improves CRISPR editing accuracy without sacrificing performance

The introduction of the CRISPR-Cas9 system for genome editing has been an incredible boon for biological research. As our understanding of CRISPR technology increases, so does our awareness of the off-target editing events carried out by Cas9 nuclease. Existing CRISPR guide RNA design tools, such as CRISPR Design (MIT [1]) and CCTop (CRISPR-Cas9 Target online predictor, Heidelberg University [2]), attempt to predict off-target editing of the guide RNAs. However, these tools are limited by an incomplete understanding of the factors that cause Cas9 to cleave at incorrect sites.

Attempts have also been made to improve the specificity of the Cas9 nuclease, but these rationally designed variants have come at some cost to the on-target performance of Cas9 [3–4]. Dr Christopher Vakulskas, a research scientist at IDT, has taken a different approach to evolve the Cas9 nuclease into a more precise genome editing tool. In the recorded webinar, Reducing off-target events in CRISPR genome editing applications with a novel, high-fidelity Cas9 nuclease, Dr Vakulskas gives an overview of how off-target editing is predicted and identified, and provides approaches you can use to improve targeting accuracy. The presentation sets the stage for a discussion about the development and use of a new high-fidelity Cas9 nuclease, Alt-R® S.p. HiFi Cas9 Nuclease 3NLS, that significantly reduces off-target editing, without sacrificing on-target performance. If you are performing genome editing experiments and you are concerned about specificity, watch the recording below to learn more.



References:

  1. Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827–832.
  2. Stemmer M, Thumberger T, et al. (2015) CCTop: An intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLOS ONE, 10(4):e0124633.
  3. Slaymaker IM, Gao L, et al. (2016) Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268):84–88.
  4. Kleinstiver BP, Pattanayak V, et al. (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587):490–495.

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.


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

A recombinant Cas9 enzyme that drastically reduces CRISPR off-target effects—Learn about the Cas9 variant, Alt-R S.p. HiFi Cas9 Nuclease 3NLS, that greatly reduces off-target cutting events during CRISPR genome editing. At the same time, it maintains the high level of on-target editing efficiency as wild-type Cas9 nuclease.

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.

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.


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


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