How much crRNA do I need?

Ordering guidelines for quantities of crRNA and tracrRNA

Edit-R CRISPR-Cas9 synthetic guide RNA simplifies genome engineering applications by eliminating the time-consuming cloning of individual sgRNA expression vectors, or preparation and purification of in vitrotranscribed sgRNAs. Our novel approach includes predesigned or custom ready-to-use synthetic reagents that enable fast assessment of multiple target sites per gene, for multiple genes.

Much like the dual guide RNAs in the endogenous S. pyogenes system, the Edit-R synthetic CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) are transfected together to complex with the Cas9 nuclease and direct genome cleavage.

Experimental Considerations

There are a lot of considerations into what makes a good CRISPR-Cas9 experiment, but one question we often get is: how much crRNA and tracrRNA do I need? The answer will depend on three primary considerations:

  1. What is your experimental reaction volume (assuming cell culture)?
  2. How many experiments will you do? Consider technical replicates and multiple cell lines.
  3. How are you delivering the crRNA and tracrRNA? (transfection reagents, electroporation, etc.)

Keeping in mind that crRNA and tracrRNA should be used in equimolar amounts, the total number of experiments you can carry out for a given quantity (nmol) of each RNA is provided in Table 1. This table assumes a working concentration of 25 nM:25 nM and the use of DharmaFECT™ transfection reagents, or similar. It is recommended to work at a range of 25-50 nM to ensure efficient cutting, but the crRNA:tracrRNA working range should be empirically determined with optimization in your particular cells with use of a positive control and subsequent determination of editing efficiency.

Smaller quantities of crRNA (< 2 nmol) are available in cherry-pick libraries; simply upload a gene list or a set of catalog numbers to order 96-well arrayed, predesigned crRNAs.

For electroporation or other instrument-based delivery methods, the amount of crRNA and tracrRNA will depend on the reaction volume, number of cells, and type of Cas9 being used. You can find guidance for electroporation in our protocols using synthetic crRNA and tracrRNA with either Cas9 mRNA, expression plasmid, or Cas9 protein.

There is also an application note that uses microinjection in a zebrafish model.

Number of Experiments in Different Reaction Volumes

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

0.5

0.5

200

40

20

8

2

2

800

160

80

32

5

5

2000

400

200

80

10

10

4000

800

400

160

20

20

8000

1600

800

320

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.

Additional Resources