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.
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.
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.
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 including local installation instructions available at: http://lmweber.github.io/CrispantCal/
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.