CRISPR-Cas-Integrated LAMP

Abstract

Pathogen-specific point-of-care (PoC) diagnostic tests have become an important need in the fight against infectious diseases and epidemics in recent years. PoC diagnostic tests are designed with the following parameters in mind: rapidity, accuracy, sensitivity, specificity, and ease of use. Molecular techniques are the gold standard for pathogen detection due to their accuracy and specificity. There are various limitations in adapting molecular diagnostic methods to PoC diagnostic tests. Efforts to overcome limitations are focused on the development of integrated molecular diagnostics by utilizing the latest technologies available to create the most successful PoC diagnostic platforms. With this point of view, a new generation technology was developed by combining loop-mediated isothermal amplification (LAMP) technology with clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) technology. This integrated approach benefits from the properties of LAMP technology, namely its high efficiency, short turnaround time, and the lack of need for a complex device. It also makes use of the programmable function of CRISPR-Cas technology and the collateral cleavage activity of certain Cas proteins that allow for convenient reporter detection. Thus, this combined technology enables the development of PoC diagnostic tests with high sensitivity, specificity, and ease of use without the need for complicated devices. In this review, we discuss the advantages and limitations of the CRISPR/Cas combined LAMP technology. We review current limitations to convert CRISPR combined LAMP into pathogen-specific PoC platforms. Furthermore, we point out the need to design more useful PoC platforms using microfabrication technologies by developing strategies that overcome the limitations of this new technology, reduce its complexity, and reduce the risk of contamination.

The development of droplet-based microfluidic virus detection technology for human infectious diseases

“…19 Chen, Liao et al have also reported a ddPCR platform that achieves high sensitivity and rapidity without the need for bulky laboratory equipment. 20 The clustered regularly interspaced short palindromic repeats (CRISPR)-based LAMP technology integrated into a cluster has signicant importance in the CRISPR-based diagnostic systems, 21,22 and the development of this 23 The automated digital microuidic (DMF) system could advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicality, as well as apply to other CRISPR-related diagnostic platforms. 24 Z. Zhang et al described a simple microuidic chip for absolute DNA quantication based on the digital LAMP method.…”

“…The clustered regularly interspaced short palindromic repeats (CRISPR)-based LAMP technology integrated into a cluster has significant importance in the CRISPR-based diagnostic systems, 21,22 and the development of this technology is urgently needed to improve diagnostic accuracy. Zhang et al proposed an automated CRISPR-LAMP platform that could precisely manipulate CRISPR-LAMP droplets and perform a combined reaction with high sensitivity and specificity.…”

Point‐Of‐Care Ultra‐Portable Single‐Molecule Bioassays for One‐Health

“…One notable example is the application of CRISPRbased paper biosensing platforms, introducing an innovative approach that could significantly advance the field of pathogen detection and diagnosis. [37][38][39][40][41][42][43][44][45][46][47] In particular, the CRISPR/Cas9 system was employed to detect Mycoplasma pneumoniae (M. pneumoniae). [48] The newly developed biosensor leverages the potential of CRISPR/Cas9 for secondary recognition, a process that follows the preamplification of the target gene utilizing a dedicated set of primers.…”

“…This is to explore devices that can be as practical and useful as a glucometer [35] and can detect protein and nucleic acid markers at the singlemolecule detection limits assuring at the same time high reliability in terms of diagnostic sensitivity and diagnostic selectivity that should be ideally better than 95-99%. Hence the focus is on scrutinizing a couple of promising POCT, single-molecule technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR), [36][37][38][39][40][41][42][43][44][45][46][47][48] and the single molecule with a large transistor (SiMoT) both as single-sensor and as a 96 multiplexing array. [8,13,[49][50][51] Among others, these are technologies that can be addressed -by just changing the recognition element, for example, the capturing antibody (to target a specific antigen/protein) or the probe (to bind a specific oligonucleotide) integrated into the disposable accessory cartridge -a large number of different diseases.…”

A paper based microfluidic platform combining LAMP-CRISPR/Cas12a for fluorometric detection of nucleic acids

“…To make LAMP suitable for diagnostic applications it is necessary to boost sensitivity and specificity through additional readouts. [8], [9] , [10] Thus, strategies coupling nucleic acid amplification technologies (NAAT) with CRISPR/Cas technologies such as DETECTOR [11], SHERLOCK [12] and HOLMESv2 [13] to boost LAMP's efficacy are emerging as the next generation of robust molecular diagnostics. These CRISPR/Cas systems operate based on a guide RNA (gRNA) dependent activation of the CRISPR -associated (Cas) enzyme that unleashes strong trans-cleavage activity cleaving single stranded nucleic acids.…”