CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a

Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification. Using the CRISPR-ENHANCE platform and following the methodology detailed in this paper, nucleic acid detection for low copy numbers can be achieved in less than an hour through either a fluorescence-based detection or a lateral flow assay. The step-by-step instructions provided, in addition to describing how to perform both assays, incorporate details on a LAMP/RT-LAMP-based target amplification step to enable detection of RNA, ssDNA and dsDNA. Furthermore, a protocol for in-house expression and purification of LbCas12a using CL7/1m7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided.

Automated summary

This paper introduces a detection system called CRISPR-ENHANCE, which achieves high sensitivity in nucleic acid detection through the use of engineered crRNA and optimized conditions, even without target pre-amplification. The detailed methods provided enable rapid nucleic acid detection in less than an hour, either through fluorescence-based detection or lateral flow assay, and include instructions for target amplification and enzyme expression and purification.