Edit-R tracrRNA

Chemically synthesized trans-activating CRISPR RNA required for use with synthetic crRNA for fast and straightforward gene editing.


Edit-R tracrRNA
The synthetic crRNA:tracrRNA approach to CRISPR-Cas9 includes transfection-ready RNA components and enables fast assessment of multiple target sites per gene, for multiple genes.

**Please note** The catalog number U-002000-120 is no longer available, it has been replaced by U-002000-05. These products are identical, just a different suffix to represent the quantity in nmoles instead of micrograms.

The Edit-R tracrRNA is a chemically synthesized and HPLC-purified long RNA molecule based on the published S. pyogenes tracrRNA sequence (Jinek, 2012). The Edit-R tracrRNA has been tested for efficient editing in multiple mammalian cell types. It is required for use with synthetic Edit-R crRNA, comprised of 20 nucleotides identical to the genomic DNA target site, or protospacer, followed by the required S. pyogenes repeat sequence that interacts with the tracrRNA.

Once delivered to the cell, the crRNA:tracrRNA complex with Cas9 nuclease to generate site-specific, DNA double-strand breaks (DSBs). When DSBs are repaired through non-homologous end-joining (NHEJ), the resulting small insertions and deletions (indels) can cause nonsense mutations resulting in gene disruption to produce a functional knockout.

Highlights

The Dharmacon Edit-R CRISPR-Cas9 synthetic crRNA platform requires three components for gene editing in mammalian cells, based on the natural S. pyogenes system:

  • a plasmid or lentiviral vector expressing a mammalian codon-optimized gene sequence encoding Cas9 nuclease
  • a chemically synthesized trans-activating CRISPR RNA (tracrRNA), and
  • a chemically synthesized CRISPR RNA (crRNA) designed to the gene target site of interest.

tracrRNA is required for use with all synthetic crRNAs.

Since crRNA and tracrRNA are used in equimolar amounts, be sure to purchase enough tracrRNA to complete experiments with all gene-targeting and control crRNAs.

How much crRNA & tracrRNA do I need?

This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended crRNA:tracrRNA working concentration (25 nM:25nM) in various plate/well formats. Calculations do not account for pipetting errors.
crRNA
nmol
tracrRNA
nmol
96-well plate
100 µL reaction volume
24-well plate
500 µL reaction volume
12-well plate
1000 µL reaction volume
6-well plate
2500 µL reaction volume
2 2 800 160 80 32
5 5 2000 400 200 80
10 10 4000 800 400 160
20 20 8000 1600 800 320
  
HazardousNo
Shelf Life12 Months
Shipping ConditionAmbient
Storage Condition-20 C
Edit-R Synthetic tracrRNA (Catalog #U-002000-xx) recruits Cas9 nuclease when hybridized to a crRNA.

Cas9 nuclease is programmed by crRNA:tracrRNA to specifically cleave target DNA

Edit-R Synthetic tracrRNA (Catalog #U-002000-xx) recruits Cas9 nuclease when hybridized to a crRNA.

Once delivered to the cell, the crRNA and tracrRNA complex with Cas9 nuclease to generate site-specific, double-stranded DNA breaks (DSBs). When DNA DSBs are repaired through non-homologous end-joining (NHEJ), the resulting small insertions and deletions (indels) can cause nonsense mutations and truncation of protein products or the introduction of a stop codon to produce gene knockouts.


UPLC trace demonstrating excellent purity of Dharmacon Edit-R synthetic tracrRNA

UPLC trace demonstrating excellent purity of Dharmacon Edit-R synthetic tracrRNA

UPLC trace demonstrating excellent purity of Dharmacon Edit-R synthetic tracrRNA

UPLC (ultra-performance liquid chromatography) analysis of Edit-R tracrRNA, a >70nt purified RNA, demonstrates the high quality achieved routinely by Dharmacon 2’ACE chemical synthesis. CRISPR-Cas9 experiments using a crRNA:tracrRNA to recruit Cas9 nuclease rely on excellent purity and sequence fidelity for optimal activity. Instrument used: Acquity UPLC by Waters.


Ready-to-use synthetic crRNA and tracrRNA greatly simplify the CRISPR gene editing workflow

Less time preparing, more time experimenting

Ready-to-use synthetic crRNA and tracrRNA greatly simplify the CRISPR gene editing workflow


References

  1. M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337(6096), 816-821 (2012).