1. CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a.
- Author
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Nguyen, Long T., Gurijala, Jeevan, Rananaware, Santosh R., Pizzano, Brianna L.M., Stone, Brandon T., and Jain, Piyush K.
- Subjects
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AFFINITY chromatography , *NON-communicable diseases , *NUCLEIC acids , *CRISPRS , *SINGLE-stranded DNA , *COMMUNICABLE diseases - Abstract
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/lm7-based affinity chromatography, which has been used to achieve a high yield and purity of the enzyme in a single-step, is provided. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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