Application Notes: Low Multiplexing

Evaluation of CRISPR-Cas9 Mediated Genome Edits with 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 processes. 

Digital PCR 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. Using two separate assays, this application note highlights how the Absolute Q digital PCR platform can be used to study the efficiency of CRISPR mediated genome edits. 

The Absolute Q is the only vertically integrated, single instrument, digital PCR platform. 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, generating digital PCR results in as little as 90 minutes. 

Absolute Q for CRISPR Highlights

  • Non stochastic partitioning increases the consistency of the dPCR process
  • Analyzing over 95% of the sample ensures rare genome editing events are not missed
  • Highly repeatable partition counts maximize consistency across reactions
  • 4-colors of optical multiplexing increases flexibility to answer complex questions
  • 90 minute complete dPCR turnaround
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