CRISPR-Cas Coupled PCR: A Sensitive and Specific Tool for Genome Editing Validation and Variant Detection

CRISPR-Cas systems have become a cornerstone of genome editing due to their remarkable precision in targeting specific DNA sequences. However, verifying these edits and identifying pre-existing variations remain crucial steps in research and clinical applications. CRISPR-Cas Coupled PCR (CC-PCR) addresses this need by combining the targeted action of Cas nucleases with the amplification power of PCR.

CC-PCR Mechanism:

The CC-PCR workflow involves three key steps:

  1. Targeted DNA Cleavage: A Cas nuclease, pre-complexed with a guide RNA (gRNA) designed for the target locus, induces a double-strand break (DSB) in the genomic DNA.
  2. PCR Primer Design: PCR primers are designed to specifically flank the Cas cleavage site, ensuring amplification of the targeted region.
  3. PCR Amplification and Detection: PCR amplifies the target region, allowing edited or variant sequences to be visualized using various methods like gel electrophoresis, capillary electrophoresis, or high-resolution melt curve analysis (HRM).

Advantages of CC-PCR:

  • High Sensitivity: PCR amplification allows CC-PCR to detect even low percentages of edited or variant alleles within a sample.
  • High Specificity: The combination of Cas nuclease targeting and specific PCR primers minimizes amplification of unintended genomic regions.
  • Ease of Use: CC-PCR utilizes readily available PCR technology, making it a relatively simple and cost-effective method.
  • Versatility: CC-PCR can be adapted to detect various types of edits (insertions, deletions, SNPs) and is applicable to diverse research and clinical settings.

Applications of CC-PCR:

  • Validation of CRISPR Edits: Following CRISPR-Cas genome editing experiments, CC-PCR efficiently confirms successful edits at the targeted locus.
  • Screening for Genetic Variants: CC-PCR can screen large populations for specific genetic variants associated with diseases or other traits.
  • Genotyping Assays: By targeting specific SNPs, CC-PCR can be used for genotyping applications.
  • Mosaic Edit Detection: CC-PCR can identify mosaicism, where only a subset of cells within a population harbor the desired edit.

Applications of CRISPR/Cas9 system. CRISPR/Cas9 system has revolutionized genome engineering: its accuracy, rapidity, and affordability permit its use in a nearly limitless range of applications. Since its discovery, researchers have been using the CRISPR/Cas9 system to cure diseases, discover new treatments, and for precision medicine. It does not stop there; beyond treating human diseases, CRISPR/Cas9 is also being utilized for studying the model and non-model insects’ biology, somatic genome editing, manufacturing biofuels, and engineering better crops (rice, wheat, etc.), etc. These advances made possible by the invention of the CRISPR/Cas9 system will change the lives of people globally

Limitations and Considerations:

  • Off-Target Effects: Similar to CRISPR editing itself, CC-PCR requires careful design of gRNA and primers to minimize amplification from unintended Cas cleavage sites (off-targets).
  • Limited Throughput: While multiplex versions exist, CC-PCR typically analyzes single loci at a time, limiting its suitability for high-throughput variant detection.

Future Directions:

Advancements in CRISPR-Cas systems and PCR technologies hold promise for further enhancing CC-PCR capabilities. Integration with digital PCR could offer even greater sensitivity for rare variant detection. Additionally, exploring alternative detection methods coupled with CC-PCR might lead to more streamlined workflows and improved automation.


CC-PCR offers a valuable tool for researchers and clinicians working with CRISPR-Cas technology. Its simplicity, sensitivity, and specificity make it a powerful technique for targeted genome editing validation, variant detection, and various other applications. As CRISPR-Cas systems and PCR technologies evolve, CC-PCR is poised to remain a key player in genome manipulation and analysis. For all your CC-PCR needs, consider Gentaur, a leading provider of high-quality reagents, enzymes, and kits specifically designed for CRISPR-Cas and CC-PCR workflows. They offer expert technical support to ensure your success in utilizing this powerful technology.