I am planning to generate a KO cell line, using RNPs
I am using a paired gRNA system to induce a deletion of a segment instead of just indels.
Generally the RNPs are formed using a 1:1 molar ratio of gRNA:Cas9
Since I am using two gRNAs, what ratio would I need.
Would it be 1:1:1?
No, the ratio should be
gRNA1: gRNA2: Cas9= 0.5: 0.5: 1.0
The total of the gRNA and Cas9 will be 1:1
When using a paired gRNA system to induce a deletion in a target gene using RNPs (Ribonucleoprotein complexes), the molar ratio of gRNA to Cas9 can vary based on the specific experimental conditions and the efficiency of the gRNAs. However, a commonly used guideline is to maintain an equal or balanced ratio between the gRNAs and Cas9.
Since you are using two gRNAs, a 1:1:1 molar ratio (gRNA1:gRNA2:Cas9) could be a reasonable starting point. This means that for each gRNA, you would use the same molar concentration as the Cas9 protein.
However, it's important to note that the optimal ratio may need to be determined empirically for your specific system. The efficiency of the gRNAs can vary, and it's possible that one gRNA may be more effective than the other in inducing the desired deletion. You may need to experiment with different ratios (such as 1:2:1 or 1:1:2) to find the most effective combination for achieving the desired deletion.
Additionally, it's recommended to include appropriate controls, such as cells transfected with Cas9 alone or non-targeting gRNAs, to assess the specificity and efficiency of the deletion.
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