Please note our tracrRNA catalog numbers have changed to reflect the addition of chemical modifications to resist nuclease degradation. Modified tracrRNA is suitable for the same applications as the unmodified tracrRNA (U-002000-*) and can be used with either modified or unmodified Edit-R crRNA. These stabilizing modifications are required only for co-electroporation with Cas9 mRNA. Learn more about these changes in this featured article.

The Edit-R tracrRNA is a chemically synthesized and HPLC-purified long RNA molecule based on the optimized published S. pyogenes tracrRNA sequence (Jinek, 2012). It is chemically modified for nuclease resistance to improve performance in applications that use co-delivery with DNA-free Cas9 nuclease. The Edit-R tracrRNA 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 protein, mRNA, 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

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.

For crRNA libraries, use (# wells)*(nmol per well) to determine the approximate amount of tracrRNA required. For instance, a 100 well library at 0.5 nmol per well would require 50 nmol of tracrRNA. The bulk sizes of tracrRNA are recommended for large library projects.

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