Evaluation of CRISPR-Cas9 Mediated Genome Edits with the Absolute Q Digital PCR platform

Background

Genome editing through the use of the CRISPR/Cas9 system has become a tool which is widely used across many scientific disciplines. As more labs employ the use of CRISPR/Cas 9 to introduce customized changes to targets, it is equally critical to have a method of monitoring the success and efficiency of these gene editing processes.

Digital PCR, which provides higher precision quantification of nucleic acids, is aptly suited for the analysis of genome editing applications such as CRISPR/Cas9 mediated knock-ins and knock-outs. This is largely enabled by dPCR’s fundamental principle of absolute quantification, which provides quantification of nucleic acid targets without the need for orthogonal standard curves. This method of quantification is more consistent and more accurate, particularly for rare or low concentration targets. This application note highlights the Absolute Q digital PCR platform paired with a 2-probe assay design designed in collaboration with Integrated DNA Technologies (IDT) which was used to detect and quantify both CRISPR meditated knock in and knock outs with precision and accuracy. 

The Absolute Q is a vertically integrated digital PCR platform which enables a simplified digital PCR workflow – using a single instrument and a single consumable to deliver complete results in under 2 hours. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of the sample across each dPCR reaction every time. Unlike many available digital PCR systems, the workflow is identical to qPCR. This means every user can generate consistent and accurate digital PCR every time with minimal hands-on steps. 

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