Plan simple CRISPR reaction stoichiometry by calculating Cas nuclease and guide RNA stock volumes per reaction and for a master mix.
For Research Use Only. This planner calculates simple stoichiometry and does not optimize CRISPR conditions. Always follow manufacturer protocols and institutional guidelines.
Enter your reaction parameters and click Calculate to see results
CRISPR-Cas systems use a Cas nuclease (like Cas9 or Cas12a) guided by a short RNA molecule (gRNA or sgRNA) to target specific DNA sequences. The stoichiometry refers to the molar relationship between these components in a reaction mixture. Proper stoichiometry ensures efficient complex formation between Cas protein and guide RNA.
C₁V₁ = C₂V₂This relationship allows us to calculate how much stock solution to add to reach a target final concentration.
VCas = (Cfinal × Vrxn) / Cstock
VgRNA = (Cfinal × ratio × Vrxn) / Cstock
The gRNA:Cas molar ratio determines how many guide RNA molecules are present for each Cas protein. Common approaches include:
Equal molar amounts. Each Cas protein has one gRNA partner. May leave some Cas unbound if gRNA is limiting.
Common choice. Excess gRNA ensures complete loading of Cas protein, improving editing efficiency.
Higher ratios may be used when gRNA stability is a concern or for specific experimental conditions.
When preparing multiple reactions, a master mix saves time and reduces pipetting errors. Adding overage (typically 10%) ensures you don't run short due to:
For ribonucleoprotein (RNP) delivery, Cas and gRNA are pre-complexed before delivery. Incubation time and conditions affect complex stability.
This calculator shows “buffer” as the remaining volume. Actual buffer composition depends on your delivery method and cell type.
Ensure accurate stock concentrations. Protein concentrations can be measured by BCA or Bradford assay; RNA by UV absorbance.
If calculated volumes exceed reaction volume, you need more concentrated stocks or a larger reaction volume.
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