Reports of CRISPR-Based Assays Exhibit Gross Errors but CRISPR Offers High Specificity

Molecular diagnostics based on clustered regularly interspaced short palindromic repeats (CRISPR) enzyme systems have been the subject of intense research. CRISPR-associated (Cas) enzyme assays are easily reconfigurable to different nucleic acid target sequences, highly specific, and compatible with simple kits and microfluidic components. We are conducting studies of the basic CRISPR enzyme kinetics. We discovered that the great majority of all CRISPR enzyme kinetics studies show data that are inconsistent and which grossly violate basic rules of mass conservation and rate laws. Further, CRISPR assays which do not use pre-amplification often report limits of detection (LoD) which are difficult to reconcile with CRISPR kinetic rates. This widespread inconsistency makes it difficult to assess the potential of CRISPR as a diagnostics tool. Following up on this, we quantified the kinetics of a range of CRISPR-Cas systems and demonstrated that kinetics fundamentally assay LoD. We also performed a study of CRISPR specificity to small mutations, including single-nucleotide polymorphisms. We are currently developing assays that leverage CRISPR specificity for cancer detection.

Learning Objectives: 

1. Describe the two key reaction steps in CRISPR diagnostic assays.

2. Explain which kinetic rate parameters affect limits of detection?

3. Explain the key advantage of CRISPR diagnostic assays, aside from reconfigurability.