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3 valuable functions of a fluorescently labeled CRISPR-Cas9 tracrRNA

Let your tracrRNA help you monitor transfection or enrich transfected cells

The CRISPR-Cas9 System has been adapted for genome editing in eukaryotic cells. In cultured cells, this method involves adding 3 main components: a CRISPR RNA (crRNA), which contains the DNA-targeting protospacer sequence; trans-activating CRISPR RNA (tracrRNA), which binds Cas9 nuclease; and Cas9 nuclease, which creates blunt-ended cuts in genomic DNA that are repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways.

The Alt-R® CRISPR-Cas9 tracrRNA has previously been optimized by adjusting its length to increase editing efficiency and by adding chemical modifications to increase resistance to nucleases. IDT scientists have conducted additional experiments to determine the best fluorescent dye and dye positions within the CRISPR RNAs for labeling. Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550 is the result of these studies. The addition of a fluorescent dye to the tracrRNA does not affect ribonucleoprotein delivery or genome editing performance. Instead, the fluorescent dye makes it possible to:

  1. Monitor transfection during optimization of transfection conditions
  2. Monitor transfection when troubleshooting experiments
  3. Simplify screening of cells with CRISPR editing events using fluorescent-activated cell sorting (FACS) analysis of transfected cells (Figure 1)

For guidance in using Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550, view the associated application note.

Use FACS to enrich for edited cells using dye-labeled Alt-R CRISPR-Cas9 tracrRNA

Figure 1. Enrichment of sorted cells leads to higher editing efficiencies. HEK-293 and Jurkat cells were transfected by electroporation (Neon® electroporation system, Thermo Fisher) with 0.5 µM ribonucleoprotein (RNP: Alt-R® S.p. Cas9 Nuclease 3NLS complexed with Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550, all provided by IDT) and carrier DNA (Alt-R Cas9 Electroporation Enhancer, IDT). Cells subjected to RNP, but without electroporation, provided the background controls and were used to set the gates during fluorescent-activated cell sorting (FACS). Cells were sorted 24 hr post-electroporation, and positive cells were re-plated and grown for an additional 48 hr. A population of the cells was not sorted, but simply re-plated, to serve as the unsorted control. Genomic DNA was isolated using QuickExtract™ solution (Epicentre) after cell incubation for 72 hr. Total editing efficiency was measured using the Alt-R Genome Editing Detection Kit (a T7 endonuclease I assay, IDT).

Product focus

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

CRISPR-Cas9 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.

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


Related products—Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System recognizes an A-T rich PAM site, providing CRISPR target sites that are not available with the CRISPR-Cas9 System. The Alt-R Cpf1 reagents have been optimized for use with A.s. Cpf1 nuclease and are not interchangeable with those for the Cas9 system.

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

Additional resources

Use of fluorescently labeled Alt-R® CRISPR-Cas9 tracrRNA – ATTO™ 550—Application note: Learn how fluorescently labeled tracrRNA can be used to detect and visualize cells that have been successfully transfected with an RNP.

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: Maureen Young, PhD, is a senior 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. ATTO is a trademark of ATTO-TEC GmbH. For specific trademark and licensing information, see www.idtdna.com/trademarks.