CRISPY™ Master Mix for qPCR-based Gene Editing Quantitation

We edited the DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) gene in HEK293 cells using an All-in-one Cas9 construct, isolated genomic DNA from the edited cells, and then performed a CRISPY assay to quantify editing success using standard (yellow curve) and snapback primers (red curve, Figure 2A). We also ran a CRISPY assay on DNA isolated from cells that had not been edited, again using standard (yellow curve) and Snapback primers (red curve, Figure 2B). As expected, the standard primers provided robust amplification in both edited and unedited DNA, however the snapback primers only provided appreciable amplification in the DNA isolated from edited cells. The ΔCt between the standard and snapback primers provides a direct measurement of editing success, and the difference between the shapes of the melt curves demonstrates that a deletion is present in the edited DNA (Figure 2C, boxed area).

The CRISPY assay requires SBI's optimized CRISPY Master Mix

The CRISPY assay is a highly robust assay that requires the use of SBI's optimized CRISPY Master Mix—for example, ThermoFisher's SYBR Green master mix will amplify both edited (Figure 3A) and wildtype (Figure 3B) templates and provides indistinguishable melt curves (Figure 3C).

The CRISPY assay delivers excellent quantitation when performed directly on cells

To demonstrate the robustness of the CRISPY assay when using cells, we designed a new set of primers to assess the success of editing the same cells used in Figure 2. The ΔCt between standard and snapback primers in a CRISPY assay done directly on cells (Figure 4A) is virtually identical to the ΔCt in a CRISPY assay done on genomic DNA isolated from the same cells (Figure 4B), demonstrating that direct amplification of cells mirrors the results from purified DNA. This finding is also supported by comparing the melt curves performed on cells (Figure 4C) and isolated DNA (Figure 4D).