Sometimes dead cells are a good thing! Universal cell death controls for CRISPR-Cas9 optimization.
Appropriate controls are essential for good experimental design. Since gene editing requires delivery of the CRISPR-Cas9 components to generate double-strand breaks as well as imperfect repair by endogenous mechanisms of the cell, effective negative and positive controls are critical for achieving efficient gene knockout and interpreting results. Non-targeting controls enable assessment of potential non-specific effects, while positive controls can be used to assess the efficiency of gene editing and serve as an indicator of good transfection in ongoing experiments.
Positive controls to optimize and monitor delivery efficiency
Delivery of CRISPR-Cas9 components to cells is relatively fast, occurring within hours. However, the development of a pronounced phenotype due to target gene knockout can take days. It is beneficial to a researcher to have a degree of certainty – earlier in the process – that the experiment has been successful, at least from the standpoint of delivery efficiency. This is particularly useful if the measured phenotype has a longer incubation time. While initial experimental optimization should be performed using a target-specific positive control in which editing efficiency can be quantitatively assessed, during many experiments a rapid indication of delivery efficiency can save resources, labor, reagents, and time, that may otherwise be wasted on performing costly and time-consuming phenotypic assays on failed experiments or interpretation of weak results due to poor delivery.
The value of a lethal control
Edit-R Lethal crRNA controls are universal positive controls based on cell death, such that cell viability can be correlated to the efficiency of the delivery of CRISPR-Cas9 components to the cells. They provide quick, visual, and quantifiable indication of transfection efficiency.
Why use Edit-R lethal controls:
- No complicated or costly phenotypic assays – the efficiency of editing can be estimated by visual inspection via microscopy or a simple cell viability assay
- Save time and resources – Determine right away whether your transfection was efficient, since gene-specific phenotypes may take longer to develop.
- Robust and consistent – Monitor the overall efficiency of delivery across multiple plates in a single screen, or a series of separate experiments
- Easy to implement – just include lethal controls alongside your experimental crRNAs, or add to your Cherry-pick plate at the time of ordering
- Compatible with different sources of Cas9 nuclease - whether you use Cas9-integrated cell lines, Cas9 protein, or Cas9 mRNA, simply deliver lethal controls to cells as you would any other crRNA.
- May be used as a sole positive control in rare cases when an appropriate assay-specific positive control is not available
- Species-specific sgRNAs targeting well-characterized genes, as well as mismatch detection assay primers, to determine the effectiveness of your gene editing conditions for maximal efficiency.
- Species-specific crRNAs targeting well-characterized genes, as well as mismatch detection assay primers.
- A convenient table to help you choose effective controls for your gene editing experiment
- Whether your goal is gene knockout or creating a knock-in, this guide will assist you in selecting the right Edit-R tools for your genome engineering application.
- A protocol for successfully using Edit-R synthetic sgRNA positive controls.
- Application note for using Edit-R synthetic crRNA positive controls for transfection optimization and ongoing monitoring of CRISPR-Cas9 experimental conditions.