The Ed-digital PCR test directly informs on the presence and number of transgenes copies inserted in the genome of CRISPR-edited cells
Thanks to its flexibility and ability to achieve precise targeting, the CRISPR system is a powerful gene editing tool for various molecular biology applications. Precise control of the transgene integration is a major challenge for human cell research biosafety. Despite the improvement of CRISPR specificity, a significant proportion of transgenes integrates at unintended sites. These off-target insertions can cause genomic alterations that may disrupt normal cell functions with dramatic consequences on research outcomes. Therefore, it is crucial to verify the transgene presence at the desired genome site using PCR or sequencing, and also to confirm the absence of any possible additional unwanted insertion using Ed-digital, Stem Genomics digital PCR test for transgene copy number quantification.
The Ed-digital test allows the detection of the number of transgene copies present in the genome of gene-edited cells using droplet digital PCR. Ed-digital targets common resistance cassettes (puromycin, neomycin) as well as the fluorescent reporter gene mCherry.
Examples of Ed-digital applications in gene-edited induced pluripotent stem cell (iPSC) lines
Analysis of transgene integration in iPSC lines (graph on the left):
A. Determination of the number of neomycin gene copies in two iPSC samples. The first sample carries two copies and the second one single copy of the transgene.
B. Determination of puromycin gene copies in two iPSC samples. The first one carries two copies and the other one displays mosaic integration (n=2.5) of the transgene.