CrispantCal: CRISPR-Cas9 Injection Mix Calculator

gRNA sample properties

gRNA molar mass: Enter DNA template sequence to calculate molar mass, or enter custom value directly. DNA sequence (e.g. ATCG)
Molar mass (g/mol)
gRNA concentration: Mass concentration (ng/µL)

Optional: Second gRNA sample

Select whether to include a second gRNA sample. If yes, enter sample properties below.
gRNA molar mass: Enter DNA template sequence to calculate molar mass, or enter custom value directly. DNA sequence (e.g. ATCG)
Molar mass (g/mol)
gRNA concentration: Mass concentration (ng/µL)

Cas9 sample properties

Choose one of the preset defaults or enter custom values.
Molar mass (kg/mol or kDa)
Mass concentration (g/L)
Volume of Cas9 solution (µL)

Total volume

Total volume of injection mix (µL)

Optional: Final Cas9 concentration

Select whether to specify final concentration of Cas9 instead of total volume. If yes, enter concentration.
Final mass concentration of Cas9 in injection mix (ng/µL)

Optional: KCl diluent

Select whether to add additional KCl diluent. If yes, enter concentrations.
KCl concentration of Cas9 solution (mM)
KCl concentration of diluent (mM)
Desired KCl concentration (mM)

Optimal injection mix


This tool calculates volumes corresponding to an optimal one-to-one molecular ratio of gRNA to Cas9 in a CRISPR-Cas9 injection mix.

Enter the molecular properties of your gRNA and Cas9 samples, volume of Cas9 solution, and desired total volume of injection mix in the input fields. The calculated volumes for the optimal mix ratio are then shown in the table and plot above.


gRNA inputs

The gRNA molar mass can be calculated by entering the DNA template sequence (e.g. ATCG...) or entering a custom value directly. If entering a DNA sequence, it can be either the full sequence (crRNA + tracrRNA, around 100 nt) or the crRNA only (around 20 nt). If you enter the crRNA only, select the option to add the tracrRNA and UUUUU-end (25,981.5 g/mol) to give the full sequence.

The calculation formula for the gRNA molar mass is: (A * 329.21) + (U * 306.17) + (C * 305.18) + (G * 345.21) + 159.0, where A, U, C, and G are the number of each nucleotide, all T's are assumed to be converted to U's, and the 159.0 term is for a 5' triphosphate. Link to formula source and further details.

The optional inputs for a second gRNA sample allow you to calculate volumes for an experiment targeting two DNA locations. The calculations assume that the two gRNAs are required in equal proportions. Output volumes are then calculated for a one-to-one ratio of Cas9 to the combined concentration of the two gRNA samples.

Cas9 inputs

Default values for Cas9 sample properties are available for the Cas9 samples currently used in the Mosimann lab at the University of Zurich.

The optional input for final Cas9 concentration allows you to specify a desired final concentration of Cas9 in the injection mix, instead of total volume.

KCl diluent

The KCl diluent option allows you to calculate the additional volume of KCl diluent required to increase the KCl concentration in the injection mix to a desired value. This option was added because optimal reaction efficiency has been observed at a KCl concentration of around 300 mM, but Cas9 samples are often provided in 100-150 mM KCl solution, which is then further diluted by the rest of the injection mix.

Additional information

Additional information including local installation instructions available at:


This tool was developed by Lukas M. Weber, Jonas Zaugg, Anastasia Felker, and Christian Mosimann (Mark D. Robinson and Christian Mosimann labs), Institute of Molecular Life Sciences, University of Zurich, Switzerland.