Two non-targeting control formats: synthetic crRNA and lentiviral sgRNA

Why are negative controls so important?

In every experiment, a negative control can help you separate the effects of the experimental variable from the overall experimental conditions. With CRISPR-Cas9 mediated gene editing, creating a high-quality non-targeting control is not simple. The easiest negative control to perform is “untreated” in which cells receive no manipulations, but this does not tell you everything. Better controls use all experimental manipulations, but with non-targeting versions of guide RNAs. Random or scrambled gRNA sequences are poor controls, as they are likely to target one or more sites and could result in a phenotypic effect due to unintended editing activity.

What makes a good negative control for CRISPR-CAS9 gene editing?

A synthetic (crRNA) non-targeting control should have two qualities:

  1. low sequence similarity of guide RNA to the genome being targeted. Studies have shown that a minimum of 3 mismatches or gaps are needed to almost completely ensure no editing will occur.
  2. contain a constant region that allows pairing with tracrRNA so that Cas9 nuclease can be loaded. In this way, the negative control is mechanistically as close as possible to the use of experimental and positive control crRNAs.

Non-targeting sgRNA controls based in lentiviral vectors should employ the same vector backbone and promoter and – similar to synthetic crRNA - contain only designs bioinformatically determined to have at least 3 or more mismatches or gaps with the experimental genome.


If your CRISPR-Cas9 experiment depends on a phenotypic measurement, the use of one or even several non-targeting controls is absolutely essential for the proper interpretation of your results! You can depend on Dharmacon CRISPR-Cas9 non-targeting controls with 5 to 10 bioinformatically designs confirmed to have 3 or more mismatches for human, mouse and rat genomes.

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