CRISPR-Based Diagnostics: Gross Errors, Useful Specificity, and Microfluidic Assays

Molecular diagnostics based on clustered regularly interspaced short palindromic repeats (CRISPR) enzyme systems have recently been the subject of intense research, development, and investment. CRISPR-associated (Cas) enzyme assays are easily reconfigurable, highly specific, and compatible with lyophilization, simple kits, and microfluidic components. We are conducting studies of the basic kinetics of CRISPR enzymes. In 2020, we discovered that the great majority of all CRISPR enzyme kinetics studies showed data that are inconsistent and grossly violate basic rules of mass conservation and rate laws. We used dimensional analysis and scaling laws to demonstrate these inconsistencies. Despite this, the last 5 years have seen a persistence of such grossly incorrect reports and, more recently, there has been an explosion of peer-reviewed publications claiming limits of detection which are extremely difficult to reconcile given existing knowledge of fundamental limits of CRISPR-based assays. We are also evaluating and applying CRISPR’s specificity and developing microfluidic assays. We performed a study of CRISPR specificity to small mutations, including single-nucleotide polymorphisms. We will also briefly present on microfluidic CRISPR assays and report on novel CRISPR assays using on-chip electric field control with a method called isotachophoresis (ITP).