Abstract

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

Juan Santiago, Professor of Mechanical Engineering, Stanford University

Molecular diagnostics based on clustered regularly interspaced short palindromic repeats (CRISPR) enzyme systems have been the subject of intense research and development. CRISPR-associated (Cas) enzyme assays are easily reconfigurable to different nucleic acid targets, 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. 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 how these kinetics fundamentally limit detection sensitivity. 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. We will also present a review of microfluidic CRISPR assays and report on our CRISPR assays using on-chip electric field control with a method called isotachophoresis (ITP).