Some CRISPR-based diagnostic assays combine RPA with CRISPR/Cas to detect nucleic acids. This apparently gives the assay an advantage by providing two rounds of specificity: primer-specific isothermal amplification (RPA), and crRNA-specific detection (CRISPR/Cas). But RPA alone also provides two rounds of specificity by using probe-specific detection. So how exactly does leveraging the assay with CRISPR/Cas confer a specificity advantage? Are there other advantages beyond ~increased specificity? Thanks in advance for any insights.
It may be too late to add to this question, but here's my take on it.
Absolutely, when CRISPR is integrated with isothermal amplifications, we're essentially incorporating an additional layer of specificity. This is especially beneficial for assays like LAMP, which, despite their remarkable sensitivity, occasionally grapple with specificity issues.
An interesting facet to consider is that the isothermal reactions in this scenario don't necessarily require meticulous optimization, as CRISPR provides a robust buffer. This can somewhat alleviate the burdens when working with RPA or LAMP. However, bear in mind that this doesn't imply a complete elimination of optimization necessities, but rather a diminished effort to achieve stellar results. A potential downside to this approach is that optimization could become more challenging when trying to merge both pre-amplification and CRISPR in a single-tube reaction, given that some components of the pre-amplification reaction could interfere with the CRISPR reaction. This is an area of ongoing exploration, with numerous intriguing studies endeavoring to tackle this complication.
Now, what I consider the most relevant advantage in the context of CRISPR-based diagnostics employing Cas12 or Cas13 with pre-amplification, is the the bonus signal amplification from the enzymes' collateral cleavage. Picture this with Cas13 for example: a single cut on the target can spark off multiple cuts of the reporter molecule. This one-to-many relationship not only boosts the specificity through CRISPR's pin-point recognition mechanism, but also jacks up the sensitivity.
However, the real goal here is to uncouple CRISPR detection from any need for target pre-amplification and still pull off results that match or even surpass those from isothermal or PCR methods. And guess what? That's exactly where recent advancements are steering us towards. Exciting times ahead!
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays